CN1930292A - Proteins imparting boron-tolerance and genes thereof - Google Patents

Proteins imparting boron-tolerance and genes thereof Download PDF

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CN1930292A
CN1930292A CNA2005800073054A CN200580007305A CN1930292A CN 1930292 A CN1930292 A CN 1930292A CN A2005800073054 A CNA2005800073054 A CN A2005800073054A CN 200580007305 A CN200580007305 A CN 200580007305A CN 1930292 A CN1930292 A CN 1930292A
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CN1930292B (en
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藤原彻
野泽彰
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Japan Science and Technology Agency
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Abstract

The present invention is to provide genes and proteins having possibilities to generate plants having tolerance against excessive boron, which can confer a boric acid tolerance to organisms. 5 types of genes that can confer a boric acid tolerance to yeast, that is, AtPAB2, AtRBP47c', AtRPS20B, AtMYB13 and AtMYB68, AtRBP45a, AtRBP45b, AtRBP45c, AtRBP45d, AtRBP47a, AtRBP47b, AtRBP47c, AtUBP1a, AtUBP1b and AtUBP1c were found by expressing several genes of higher plant Arabidopsis thaliana in yeast that is a organism model of eukaryote. Further, it was found that a key of the toxicity mechanism of boric acid exists in the specific inhibition of splicing, and a gene related to enhancement of splicing efficiency is a gene that confers a boric acid tolerance.

Description

Proteins imparting boron-tolerance and gene thereof
Technical field
The present invention relates to the screening method that a kind of Arabidopis thaliana mesoboric acid resistance gives protein and gene thereof, contains the recombinant vectors of this gene, the transformant that has imported this recombinant vectors and imparting boron-tolerance gene.
Background technology
Boron is a kind of (for example, non-patent literature 1 with reference to) in the necessary trace element of higher plant.Boron is also toxic, if superfluous picked-up can be restrained growth and development of plant, perhaps animal also can be dead owing to acute poisoning.Boron in the soil solution exists with uncharged molecularity.Therefore than being easier to elimination, deficiency disease takes place easily in farm crop.Report that now in the country of the whole world more than 80 that comprises Japan, 130 kinds are because boron is owed output on the agricultural that weary disease causes, the decline of quality (for example, with reference to non-patent literature 2).In addition, known boron is narrower than the optimum concentration range of other element, it is little with the concentration difference that manifests excessive symptom to demonstrate the concentration of owing weary symptom.Therefore, on agricultural, be difficult to regulate the rate of fertilizer application of boron always.Especially, when having executed the boron of excess quantity, be difficult to remove boron, when the crop production in this farmland, will bring obstacle.In addition, owing to contain boron in the water channel water, the harm of boron excess is many when therefore carrying out agro-farming in dry area by irrigation can form problem.So, surplus has applied the field of boron and high regional widely distributed of boron concentration in the world, each country that comprises these areas just with at the countermeasure of the harm of boron excess as the important topic on the agricultural policy.In addition, boron also is present in metal conditioner and the SYNTHETIC OPTICAL WHITNER, contains a large amount of boron from the water that the factory that uses these materials discharges.Among the mankind, the lethal quantity of boron is 15-20g, yet even known boron than this lethal quantity less amount also can cause various damages in digestion organs and neural system.Now, contained boron amount has now become problem in the industrial water drainage.
Recently, gradually known the effect of the boron in the plant.Known the pectin substance polysaccharide (for example, with reference to non-patent literature 3) of the crosslinked cell walls of boron.Also disclose this crosslinked be growth and development of plants necessary (for example, non-patent literature 4 with reference to).The viewpoint of molecular level the earliest that relates to the physiological function of boron in Here it is the plant.On the one hand, still residual a lot of unclear places in the intravital boron conveyer mechanism of plant.Think that always boron by secular passive the diffusing in the cell of bilayer lipid membrane, is transported to (for example, with reference to non-patent literature 5) in the plant materials by transpiration stream.On the other hand, the known boron nutrition condition that is suitable for growing is very big with the product difference between species between planting.As the possibility of its reason, what can enumerate is to absorb and transposition, the difference of utilising efficiency, but and unclear molecule as its key element.In recent years, proposed to carry (for example, with reference to non-patent literature 6), but its experiment in vitro, do not demonstrated in the individuality of reality these vessel elements and whether carry relevant with boron according to expression system in the ovocyte that only uses Africa xenopus and membrane vesicle by conduit.In addition, though have the energy transport (for example) of being undertaken, also do not identify the vehicle of bearing this effect by vehicle with reference to non-patent literature 7 according to the root absorption experiment prompting of Sunflower Receptacle.
The inventor isolates discharge type boron resistance protein BOR1 (for example, with reference to patent documentation 1) first from the model plant Arabidopis thaliana in organic sphere.It is generally acknowledged that BOR1 bears the effect (for example, with reference to non-patent literature 8) that conductive pipe is carried boron energetically under low boron nutrition condition.In addition, as the resistance of bearing boron conveying effect, except BOR1, earlier known zymic YNL275w (for example, with reference to non-patent literature 9) in addition.
In addition, as mentioned above, boron (B) is the necessary micro-nutrients of plant (for example, non-patent literature 10 with reference to) and animal (for example, non-patent literature 11 with reference to), but under the high density is deleterious (for example, with reference to non-patent literature 12,13).The SOIL DISTRIBUTION that contains spontaneous high density boron is in all over the world, and the mankind's activities such as irrigation of the water of the fertilising of boron, fossil-fueled, use boracic have caused the environment of high density boron (for example, with reference to non-patent literature 12,13).
As in the plant because the symptom that the toxicity of boron produces, comprise the yellows (for example) of leaf margin, the unusual and/or bark downright bad (for example, with reference to non-patent literature 14) of fruit with reference to non-patent literature 13.The boron of excess quantity reduces output of crop and quality.The toxicity of boron has now become the great obstacle of agriculture production in the world.Boron is poisonous to animal and microorganism with high density.The lethal quantity that can infer boron is about 140mg/kg in the adult, is about 270mg/kg (for example, with reference to non-patent literature 15,16) in the child.If through the boron of long-term picked-up high density, will in human body, bring out poor appetite, nauseating, the minimizing of body weight, go down (for example, with reference to the non-patent literature 17) of sexual desire.Now, shown that the safe intake of the boron that can allow is for being grown up 13mg/ day (for example, with reference to non-patent literature 18).Because boron has sterilization effect to microorganism, so comprises boron gradually in the sanitas of food (for example, with reference to non-patent literature 19).And then boron is as long-term sterilant, and it is used in particularly cockroach sterilant (for example, with reference to non-patent literature 20) gradually.
Recent decades, since the toxicity of recognizing boron, just begun to have carried out to be used to study the research of the toxic influence of boron.Wherein many is physiological research.For example, in the leaf of soybean, the allantoic acid acid amides adds the activity of water decomposition enzyme owing to boric acid reduces (for example, with reference to non-patent literature 21).In the coral stonewort (Chara corallina), observe because the inhibition to malate dehydrogenase (malic acid dehydrogenase) and isolemon dehydrogenase activity (for example, with reference to non-patent literature 22) that boron produces.In addition, boron amount in the placenta and the active negative correlation (for example, with reference to non-patent literature 23) of Δ aminolevulinate dehydratase that relates among porphobilinogen (porphobilinogen) (porphyrin synthetic medium) the synthetic newborn infant have also been reported.
We think that can dissolve borate plays an important role to the toxicity of boron.Part is converted into borate to intracellular boric acid because internal pH is high.If high concentration of boric acid is provided for cell, borate concentration rises in the cell, forms the borate complex body with the various cis-diol that comprise intracellular molecule.This cis-diol that comprises molecule comprises NAD+, ATP, S-Ado Met, RNA and several sugar (for example, with reference to non-patent literature 24,25).These molecules owing to can be used as the coenzyme and/or the matrix of plurality of enzymes, may be induced boratory combination, the disappearance of function or the reorganization of enzymic activity, the inhibition of biochemical reaction and final metabolic disturbance.Though accumulated biological chemistry and physiological analysis, and about the supposition of the toxic effect of boron, the toxicity molecule mechanism of the boron of inducing cell death is not clear.
[patent documentation 1]
The spy opens the 2002-262872 communique
[non-patent literature 1]
Loomis，W.D.；Durst，R.W.(1992)Chemistry?and?biology?of?boron.Biofactors?3：229-239
[non-patent literature 2]
Shorrocks，V.M.(1997)The?occurrence?and?correction?of?boron?deficiency.Plant?and?Soil?193：121-148
[non-patent literature 3]
Matoh，T.；Ishigaki，K.I.；Ohno，K.；Azuma，J.I.(1993)Isolation?and?characterization?of?aboron-polysaccharide?complex?from?radish?roots.Plant?Cell?Physiol.34：639-642
[non-patent literature 4]
O′Neill，M.A.；Eberhard，S.；Albersheim，P.；Darvill，A.G.(2001)Requirement?of?boratecross-linking?of?cell?wall?rhamnogalacturonan?II?for?Arabidopsis?growth.Science?294：846-849
[non-patent literature 5]
Marschner，H.(1995)Mineral?Nutritin?of?Higher?Plants，2nd?ed.Academic?Press，San?Diego，CA
[non-patent literature 6]
Dordas，C.；Chrispeels，M.J.；Brown，P.H.(2000)Permeability?and?channel-mediatedtransport?of?boric?acid?across?membrane?vesicles?isolated?from?Squash?roots.Plant?Physiol.124：1349-1362
[non-patent literature 7]
Dannel，F.；Heidrun，P；Romheld，V.(2000)Characterization?of?root?boron?pools，boron?uptakeand?boron?translocation?in?sunflower?using?the?stable?isotope?10B?and?11B.Aust.J.Plant?Physiol.156：756-761
[non-patent literature 8]
Takano，J.；Noguchi，K.；Yasumori，M.；Kobayashi，M.；Gajdos，Z.；Miwa，K.；Hayashi，H.；Yoneyama，T.；Fujiwara，T.(2002)Arabidopsis?boron?transporter?for?xylem?loading.Nature?420(6913)：337-340
[non-patent literature 9]
Zhao，R.M.；Reithmeier，R.A.F.(2001)Expression?and?characterization?of?the?aniontransporter?homologue?YNL275w?in?Saccharomyces?cerevisiae.American?Journal?ofPhysiology-Cell?Physiology?281(1)：C33-C45
[non-patent literature 10]
Warington，K.(1923)Ann.Bot.37，629-672.
[non-patent literature 11]
Park，M.，Li，Q.，Shcheynikov，N.，Zeng，W.，&?Muallern，S.(2004)Mol.Cell?16，331-341.
[non-patent literature 12]
Gupta，U.C.，Jame，Y.W.，Campbell，C.A.，Leyshon，A.J.，&?Nicholaichuk，W.(1985)Can.J.Soil?Sci.65，381-409.
[non-patent literature 13]
Nable，R.O.，Banuelos，G.S.，&?Paull，J.G.(1997)Plant?Soil?193，181-198.
[non-patent literature 14]
Brown，P.H.，&?Hu，H.(1996).Ann.Bot.77，497-505.
[non-patent literature 15]
Young，E.G.，Smith，R.P.，&?MacIntosh，O.C.(1949)Can.Med.Assoc.J.61.447-450.
[non-patent literature 16]
Arena，J.M.，&?Drew，R.H.(1986)in?Poisoning，(C.C.Thomas，Splingfield).pp.131.
[non-patent literature 17]
Hunt，C.D.(1993)in?Encyclopedia?of?Food?Science，Food?Technology?and?Nutrition，vol.1，eds.Macrae，R.，Robinson，R.K.&?Sadler，MJ.(Academic?Press，London)，pp?440-447.
[non-patent literature 18]
WHO/FAO/IAEA(1996)in?Trace?Elements?in?Human?Nutrition?and?Health，(World?HealthOrganization，Geneva)，pp.175-179.
[non-patent literature 19]
Nielsen，F.H.(1997)Plant?Soil?193，199-208.
[non-patent literature 20]
Cochran，D.G.(1995)Experientia51，561-563.
[non-patent literature 21]
Lukaszewski，K.M.，Blevins，D.G.，&?Randall，D.D.(1992)Plant?Physiol.99，1670-1676.
[non-patent literature 22]
Reid?R.J.，Hayes?J.E.，Post?A.，Stangoulis?J.C.R.，&?Graham?R.D.(2004)Plant?Cell?Environ.27，1405-1414.
[non-patent literature 23]
Huel，G.，Yazbeck，C.，Burnel，D.，Missy，P.，&?Kloppmann.W.(2004)Toxicol.Sci.80，304-309.
[non-patent literature 24]
Ralston，N.V.C.，&?Hunt，C.D.(2000)FASEB?J.14，A538.
[non-patent literature 25]
Ricardo，A.，Carrigan，M.A.，Olcott，A.N.，&?Benner，S.A.(2004)Science?303，196.
Summary of the invention
The technical problem to be solved in the present invention
Give gene by import yeast mesoboric acid resistance plant, just have and to prepare the plant possibility that boron excess is had resistance.We think that the boron resistance plant is of value to the crop yield of the region that is subjected to boron excess harm.In addition, also there are algae and bacterium by using the boric acid resistance that imports these genes to improve, also can be by making it absorb boron contained in process water to remove the possibility of using in the environmental purification such as boron.The technical problem to be solved in the present invention is to provide and can prepares gene that gives biological boric acid resistance and the protein that boron excess is had the plant of resistance.In addition, also be to provide the method for efficient screening imparting boron-tolerance gene by understanding the toxic mechanism of boric acid.
The method that is used for the technical solution problem
The inventor has carried out positive research in order to solve the problems of the technologies described above, express in Eukaryotic model animals yeast by the several genes that make the higher plant Arabidopis thaliana, 5 kinds of genes of yeast boric acid resistance have been found to give, be AtPAB2, AtRBP47, AtRPS20B, AtMYB13, and each gene of AtMYB68, finish thereby finished the present invention.In addition, the key of the toxic mechanism of boric acid is the specificity of montage is suppressed, and finds that the gene relevant with the enhancing of montage efficient is the imparting boron-tolerance gene, thereby finishes the present invention.
That is to say, the present invention relates to (1) coding by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have the active protein DNA of imparting boron-tolerance and, (2) coding is by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the protein of the formation of the aminoacid sequence shown in 30, and have the active DNA of imparting boron-tolerance and, (3) by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27, or the imparting boron-tolerance gene DNA that constitutes of the base sequence shown in 29 or its complementary sequence and, (4) are by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27, or lack in the base sequence shown in 29, replace or added that the base sequence of 1 or several bases constitutes and coding have the active protein DNA of imparting boron-tolerance and, (5) hybridize under stringent condition with the DNA of claim 3 record and encode have the active protein DNA of imparting boron-tolerance with, (6) are by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have the active protein of imparting boron-tolerance and, (7) are by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or lack in the aminoacid sequence shown in 30, replace or added that 1 or several amino acid whose aminoacid sequences constitute, and have the active protein of imparting boron-tolerance and, (8) contain the DNA of each record of claim 1～5, and can express proteins imparting boron-tolerance recombinant vectors and, (9) imported the recombinant vectors that claim 8 is put down in writing, and express the proteins imparting boron-tolerance transformant and, the transformant of (10) claim 9 record, it is characterized in that transformant be yeast and, (11) transformant of claim 9 record, it is characterized in that transformant be plant and, the screening method of (12) imparting boron-tolerance gene is characterized in that using gene library, disappearance YNL275w gene, transform the YNL275w do not express YNL275w and destroy yeast, in containing the substratum of boric acid, cultivate gained and transform YNL275w and destroy yeast, measure and estimate this conversions YNL275w destroy the zymic imparting boron-tolerance active with, (13) screening method of imparting boron-tolerance gene, it is characterized in that suppressing with the specificity to montage that is produced by boric acid is target, measure and estimate montage efficient the enhancing degree and, the screening method of the imparting boron-tolerance gene of (14) claim 13 record, it is characterized in that making analyte in yeast cell, to express, in the presence of boric acid, cultivate analyte and express, the improvement degree that suppresses with the specificity that produces by boric acid of the gene that contains intron in the yeast to montage measure and estimate as the enhancing degree of montage efficient and, (15) screening method of the imparting boron-tolerance gene of claim 14 record, the gene that it is characterized in that containing in the yeast intron be in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genome the RPL7B gene and, the DNA of (16) each record of claim 1～5 as the purposes of imparting boron-tolerance gene and, the DNA of (17) each record of claim 1～5 be used to prepare the plant that has been given the boric acid resistance or zymic purposes and, (18) claim 6 or 7 the record protein as have the active proteinic purposes of imparting boron-tolerance and, (19) claim 6 or 7 the record protein be used to prepare plant or the zymic purposes that has been given the boric acid resistance.
The effect of invention
By in plant, importing imparting boron-tolerance gene of the present invention, just exist and to prepare the plant possibility that boron excess is had resistance.We think that the boron resistance plant is of value to the crop yield of the region that is subjected to boron excess harm.In addition, also there are algae and bacterium by using the boric acid resistance that imports these genes to improve, also can be by making it absorb boron contained in process water to remove the possibility of using in the environmental purification such as boron.
Embodiment
As gene DNA of the present invention, if (A) coding is arranged by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have an active protein DNA of imparting boron-tolerance; (B) coding is by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute and have an active protein DNA of imparting boron-tolerance; (C) by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27, or the imparting boron-tolerance gene DNA imparting boron-tolerance gene DNA of the base sequence shown in 29 or its complementary sequence formation (the Arabidopis thaliana source); (D), or disappearance in the base sequence shown in 29, replace or added that the base sequence of 1 or several bases constitutes and coding has the active protein DNA of imparting boron-tolerance by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27; Or (E) and by sequence number 1,3,5,7,9,11,13.15,17,19,21,23,25,27, or the imparting boron-tolerance gene DNA that constitutes of the base sequence shown in 29 is hybridized under stringent condition and is encoded and has the active protein DNA of imparting boron-tolerance; The imparting boron-tolerance gene that constitutes has no particular limits.In addition, as proteins imparting boron-tolerance of the present invention, if (A) by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have the active protein of imparting boron-tolerance; Or (B) by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or disappearance in the aminoacid sequence shown in 30, replace or added that the amino acid whose aminoacid sequences of 1 or several constitute, and have the active protein of imparting boron-tolerance; Have no particular limits, wherein so-called " imparting boron-tolerance gene " is meant the gene that gives organism boric acid resistance, and so-called " proteins imparting boron-tolerance " is meant the protein that gives organism boric acid resistance.
Above-mentioned what is called " has the active protein of imparting boron-tolerance " and is meant the protein to the resistance of boric acid such as the organism that gives yeast, plant, even this proteinic plant of high expression level and yeast also can grow in the presence of high concentration of boric acid.
Imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 1, can exemplify the AtPAB2 gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 2, can exemplify AtPAB2, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 3, can exemplify AtRBP47c ' gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 4, can exemplify AtRBP47c ', imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 5, can exemplify the AtRPS20B gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 6, can exemplify AtRPS20B, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 7, can exemplify the AtMYB13 gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 8, can exemplify AtMYB13, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 9, can exemplify the AtMYB68 gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 10, can exemplify AtMYB68, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 11, can exemplify the AtRBP45a gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 12, can exemplify AtRBP45a, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 13, can exemplify the AtRBP45b gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 14, can exemplify AtRBP45b, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 15, can exemplify the AtRBP45c gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 16, can exemplify AtRBP45c, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 17, can exemplify the AtRBP45d gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 18, can exemplify AtRBP45d, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 19, can exemplify the AtRBP47a gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 20, can exemplify AtRBP47a, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 21, can exemplify the AtRBP47b gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 22, can exemplify AtRBP47b, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 23, can exemplify the AtRBP47c gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 24, can exemplify AtRBP47c, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 25, can exemplify the AtUBP1a gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 26, can exemplify AtUBP1a, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 27, can exemplify the AtUBP1b gene, proteins imparting boron-tolerance as the formation of the aminoacid sequence shown in the sequence number 28, can exemplify AtUBP1b, imparting boron-tolerance gene as the formation of the base sequence shown in the sequence number 29, can exemplify the AtUBP1c gene, proteins imparting boron-tolerance as the aminoacid sequence shown in the sequence number 30 constitutes can exemplify AtUBP1c.
Above-mentioned what is called " has lacked, has replaced or added 1 or several amino acid whose aminoacid sequences " and for example be meant and lacked, replaced or added 1～20, preferred 1～15, more preferably 1～10, the more preferably amino acid whose aminoacid sequence of 1～5 any number.In addition, above-mentioned what is called " has lacked, has replaced or added the base sequence of 1 or several bases " and for example be meant and lacked, replaced or added 1～20, preferred 1～15, more preferably 1～10, the more preferably base sequence of the base of 1～5 any number.
For example, these disappearances, replacing or added the DNA (abnormal dna) that the base sequence of 1 or several bases constitutes also can be by chemosynthesis, engineered method, and mutagenesis etc. well known to a person skilled in the art that any means prepares.Concrete is, by for by sequence number 1,3, the DNA that base sequence shown in 5,7 or 9 constitutes, adopt the method that makes it with as the medicament contact action of mutagen, the method for irradiation ultraviolet radiation, engineered methods etc. import variation in these DNA, can obtain abnormal dna.The mutagenesis in the specific specificity site in the engineered method is owing to be to import the method for specific variation thereby be useful at specific site, can be according to Molecular Cloning:A laboratoryMannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., (1989. abbreviating " molecular cloning " the 2nd edition later on as), Current Protocols in Molecular Biology, Supplement 1～38, John Wiley ﹠amp; The method of record is carried out among the Sons (1987-1997) etc.By adopting suitable expression system that abnormal dna is expressed, can obtain to lack, replace or add the protein that 1 or several amino acid whose aminoacid sequences constitute.
Above-mentioned what is called " stringent condition is the base sequence of hybridization down " is meant that nucleic acid such as using DNA or RNA is as probe, by adopting the clone hybridization method, obtainable base sequences such as plaque hybridization method or southern hybrid method, specifically can enumerate by using the DNA that fixed clone or plaque origin or the segmental filter membrane of this DNA, in the presence of the NaCl of 0.7-1.0M, after hybridizing under 65 ℃, (composition of the SSC solution of 1 times of concentration is a 150mM sodium-chlor with about 0.1-2 times SSC solution, the 15mM Trisodium Citrate), the DNA that cleaning filter membranes can be identified under 65 ℃ of conditions.Can hybridize according to the method for middle records such as " molecular cloning " the 2nd edition.
For example, as the DNA that can under stringent condition, hybridize, can enumerate the DNA that has necessarily above homology with the base sequence of the DNA that uses as probe, the preferred examples that can enumerate has and for example has more than 60% or 60%, more than preferred 70% or 70%, more preferably more than 80% or 80%, more preferably more than 90% or 90%, more than preferred especially 95% or 95%, the DNA of the homology more than 98% or 98% most preferably.
There is no particular limitation for the preparation method of gene of the present invention and preparation method, based on disclosed sequence number 1,3,5 in this specification sheets, 7, base sequence information shown in 9 or sequence number 2,4,6,8, amino acid sequence information shown in 10 prepares suitable probe and primer, exists the cDNA library of this gene can separate goal gene by screening through prediction with them, perhaps can prepare by chemosynthesis according to ordinary method.
Concrete is, prepares the cDNA library according to ordinary method from the Arabidopis thaliana that separates gene of the present invention, then, by from this library, uses the peculiar suitable probe of gene of the present invention to select desirable clone, can obtain gene of the present invention.As the origin of above-mentioned cDNA, can enumerate the various cells or the tissue of above-mentioned plant origin, in addition, separate and the total RNA of purifying, mRNA from these cell or tissues, obtaining cDNA and its clone etc. can implement according to arbitrary ordinary method.From the cDNA library method of screening gene of the present invention for example can exemplify, the method for record in " molecular cloning " the 2nd edition etc. etc., the method that those skilled in the art use always.
In addition, mutant gene of the present invention or homologous genes as above-mentioned (B)～(F) base sequence formation shown in each, can have sequence number 1 by utilization, 3,5, base sequence shown in 7 or 9 or its a part of dna fragmentation, the homologue that screens this DNA from other organism etc. under proper condition separates.In addition, can also be prepared by the preparation method of aforesaid abnormal dna.
There is no particular limitation for proteinic acquisition of the present invention and preparation method, can be the protein of natural origin, the protein of chemosynthesis, any of the recombinant protein by gene recombination technology preparation.In the proteinic situation that obtains natural origin,, can obtain protein of the present invention by appropriate combination separation and purification method of protein from express this proteinic cell or tissue.When preparing protein by chemosynthesis, according to for example, Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method chemical synthesiss such as (tertbutyloxycarbonyl methods) can be synthesized protein of the present invention.In addition, utilize the also synthetic protein of the present invention of various commercially available peptide synthesizers.When preparing protein,, can prepare protein of the present invention by in the expression system of the best, importing the DNA that constitutes by this proteinic base sequence of coding by gene recombination technology.Even in these methods, be prepared by gene recombination technology more maneuverable and that can prepare in a large number and remain preferred.
For example, pass through gene recombination technology, when preparing protein of the present invention, in order from cell culture, to reclaim this protein of purifying, can use and comprise ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography be at interior known method, preferred high performance liquid chromatography.Especially, by for example using, combine post at antibody such as proteinic monoclonal antibodies of the present invention as the post that uses in the affinity chromatography, perhaps on the protein of the invention described above, add conventional when peptide-labeled, use combines the post of peptide-labeled therewith certain material with affinity as the post that uses in the affinity chromatography, can obtain these protein purification things.In addition, protein of the present invention when endoglin expression, make the effect of cytolemma lytic enzyme after, can obtain the purifying goods by carrying out above-mentioned purification process.
And then, if those skilled in the art, according to difference presentation code sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the sequence number 1,3,5,7 of a wherein example of the base sequence of the aminoacid sequence shown in 30,9,11,13,15,17,19,21,23,25,27, or the information of the base sequence shown in 29, just can suitably prepare or obtain by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or lack in the aminoacid sequence shown in 30, replace or added the protein that 1 or several amino acid whose aminoacid sequences constitute, or with sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 has the protein of the aminoacid sequence formation of the homology more than 60% or 60%.For example, to have sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27, or the base sequence shown in 29 or its a part of DNA by screening the homologue of this DNA under suitable condition, can separate it biology beyond Arabidopis thaliana as probe.Behind the full length DNA of this homologue DNA of clone, be incorporated in the expression vector, make it in suitable host, to express, can prepare protein by this homologue dna encoding.
As recombinant vectors of the present invention, if the aforementioned recombinant vectors that contains gene of the present invention and can express proteins imparting boron-tolerance, have no particular limits recombinant vectors of the present invention, can be of the present invention gene constructed by in expression vector, suitably integrating.As expression vector, preferably can be in host cell self-replicating carrier or can be incorporated into carrier in the karyomit(e) of host cell, in addition, can bestly use the carrier that contains control sequences such as promotor, enhanser, terminator in the site that can express gene of the present invention.As expression vector, can yeast with expression vector, vegetable cell with expression vector, bacterium with expression vector, zooblast with expression vector etc., but the preferred yeast that adopts is with expression vector and the vegetable cell recombinant vectors with expression vector.
Expression vector as yeast is used can exemplify, for example, pYES2 (Invitrogen), YEp13 (ATCC37115), YEp24 (ATCC37051), Ycp5O (ATCC37419), pHS19, pHS15 etc.The promotor of using as yeast, specifically can exemplify, for example, promotor such as PHO5 promotor, PGK promotor, GAP promotor, ADH promotor, GAL1 promotor, GAL10 promotor, heat shock protein bak promoter, MF α 1 promotor, CUP1 promotor.
The expression vector of using as vegetable cell, can exemplify, for example, Ti-plasmids (tl plasmid, Tumor inducingplasmid), pSPORT1, pT7Blue-T carrier, pIG121-Hm[Plant Cell Report, 15,809-814 (1995)], pBI121[EMBO J.6,3901-3907 (1987)] etc. plasmid or tobacco mosaic virus (TMV), cauliflower mosaic virus, geminivirus infection plant viral vector such as (geminivirus) etc.The promotor of using as vegetable cell, can exemplify, for example, [Mol.Gen.Genet (1990) 220 for the cauliflower mosaic virus 35S promoter, 389-392], diphosphoribulose carboxylase small subunit promotor etc., as terminator, can exemplify for example terminator of rouge alkali synthetase gene.
In addition, as transformant of the present invention, if imported the recombinant vectors of the invention described above and expressed the transformant of proteins imparting boron-tolerance, have no particular limits, transformed yeast be can exemplify, plant (cell, tissue, individuality), transform bacteria, transformed animal (cell, tissue, individuality) transformed, but preferred transformed yeast and conversion plant (cell, tissue, individuality).
As the preparation transformed yeast is operable host's yeast, can exemplify yeast saccharomyces cerevisiae (Saccharomycescerevisae), schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyceslactis), Trichosporon pullulans (Trichosporon pullulans), riverbank and permitted prosperous yeast (Schwanniomyces alluvius) etc.Method as import from recombinant vectors to yeast host can exemplify, for example, electroporation, protoplasm body, Lithium Acetate method etc.
Operable host plant (cell, tissue, individuality) when preparation transforms plant (cell, tissue, individuality), there is no particular limitation for its kind, can be from flowers, fruit plant, edible wild herbs, root vegetables, cereal, reward leaf plant, comprise the plants such as trees of fruit tree, for example belong to Solanaceae, Gramineae, suitably select in the culturing cell of rape section, composite family, sesame section, Oleaceae, Myrtaceae, the Rosaceae, pulse family, Arecaceae or rubiaceous plant and these plants, the tissue (seed, callus etc.).In order to prepare this conversion plant, can adopt the recombinant vectors that uses the invention described above that contains gene of the present invention, in vegetable cell, import this recombinant vectors, import the method for gene DNA of the present invention in the genomic dna in vegetable cell.The conversion of plant can be according to floristics etc., suitably adopts known methods such as leaf dish coexistence culture method, electroporation, agrobacterium co-cultivation, particle bombardment to carry out.In addition, also can adopt perviousness direct acquisition recombinant vectors of the present invention in recipient cell to prepare the method that transforms plant by improve vegetable cell with physics or chemical mode.
Screening method as imparting boron-tolerance gene of the present invention, if adopt the gene library of each kind of plant, yeast etc., disappearance YNL275w gene, transform the YNL275w that does not express YNL275w and destroy yeast, contain the conversion YNL275w that cultivates acquisition in the substratum at boric acid and destroy yeast, measure and estimate this conversion YNL275w and destroy the active method of zymic imparting boron-tolerance, have no particular limits.As active mensuration of imparting boron-tolerance and evaluation, can exemplify boric acid and contain in the substratum and to transform that zymic grows and the mensuration and the evaluation of the degree of breeding.In addition, as the destruction strain of YNL275w, can the best yeast saccharomyces cerevisiae 1169 strains (Winzeler, E.A. have been exemplified; Shoemaker, D.D.; Astromoff, A.; Liang, H.; Anderson, K.; Andre, B.; Banghsm, R.; Benito, R.; Boeke, J.D.; Bussey, H.; Chu, A.M.; Connelly, C.; Davis, K.; Dietrich, F.; Dow, S.W.; El Bakkoury, M.; Foury, F.; Friend, S.H.; Gentalen, E.; Giaever, G.; Hegemann, J.H.; Jones, T.; Laub, M.; Liao, H.:Liebundguth, N.; Lockhart, D.J.; Lucau-Danila, A.; Lussier, M.; M ' Rabeet, N.; Menard, P.; Mittmann, M.; Pai, C.; Rebischung, C.; Revuelta, J.L.; Riles, L.; Roberts, C.J.; Ross-MacDonald, P.; Scherens, B.; Snyder, M.; Sookhai-Mahadeo, S.; Storms, R.K.; Veronneau, S.; Voet, M.; Volckaert, G.; Ward, T.R.; Wysocki, R.; Yen, G.S.; Yu, K.X.; Zimmermann, K.; Philippsen, P.; Johnston, M.; Davis, R.W. (1999) Functional characterization of the Saccharomyces cerevisiaegenome by gene deletion and parallel analysis.Science 285:901-906).The yeast that uses in the screening not only can use the destruction strain of YNL275w, can also use its wild-type.
In addition, screening method as imparting boron-tolerance gene of the present invention, the specificity inhibition to montage that can exemplify to be produced by boric acid is a target, measure and estimate the enhancing degree methods of montage efficient, can illustrate as follows, analyte is expressed in yeast cell, in the presence of boric acid, the cultivation analyte is expressed, and measures and estimate improvement degree that the specificity to montage that is produced by boric acid of the gene that contains intron in the yeast the suppresses enhancing degree as montage efficient.As the gene that contains intron in the yeast, specifically can illustrate the necessary proteinic gene RPL7B of ribosomal large subunit gene (sequence number 33) in the coding genes of brewing yeast group.The improvement degree that the specificity to montage that is produced by boric acid suppresses can be measured by for example RT-PCR, in addition, at this moment, preferably with imparting boron-tolerance gene A tRBP47c ' gene as positive control.
Among the present invention, comprise the DNA of the invention described above the purposes as the imparting boron-tolerance gene (method), the invention described above DNA be used to prepare the plant that has been given the boric acid resistance or zymic purposes (method) and, the proteinic conduct of the invention described above have the active proteinic purposes of imparting boron-tolerance (method) and, the proteinic of the invention described above be used to prepare plant or the zymic purposes (method) that has been given the boric acid resistance.Thereby, be included in the embodiments of the present invention and use the imparting boron-tolerance gene of the invention described above when preparation has been given the plant of boric acid resistance and yeast and have the active protein of imparting boron-tolerance (proteins imparting boron-tolerance).
Below.By the present invention will be described in more detail, but technical scope of the present invention is not subjected to the qualification of these embodiment.
Embodiment 1
[for examination yeast and plasmid]
As yeast, use yeast saccharomyces cerevisiae 1169 strains (available from Research Genetics company) and yeast saccharomyces cerevisiae BY4741 strain (available from Research Genetics company).Its genotype is respectively that 1169 strains are MATa; His3 Δ 1; Leu2 Δ 0; Met15 Δ 0; Ura3 Δ 0; YNL275w ∷ kanMX4, the BY4741 strain is MATa; His3 Δ 1; Leu2 Δ 0; Met15 Δ 0; Ura3 Δ 0.In addition, plasmid uses pYES2 (available from Invitrogen Genetics company) and pFL61 (to be provided by Nicolaus von doctor Wiren German, ホ one ヘ Application Ha イ system university; Minet M., Dufour M.-E., and Lacroute F. (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thalianacDNAs.Plant is J.2; 417-422.).Use pFL61 with preparation Arabidopis thaliana expression library.
[the boric acid resistant proofs of yeast 1169 strains]
Boric acid resistance to yeast 1169 strains used can detect.Single bacterium colony of yeast 1169 strains that transform through pYES2 and pYES2-BOR1 (downstream of the GAL1 promotor of pYES2 carrier is inserted with the carrier of CDS of the boron resistant gene BOR1 of Arabidopis thaliana) with transfering loop picking (か I と り), vibration is cultivated until OD in the SD liquid nutrient medium 600Value reach about 1.0.With nutrient solution line respectively contain 0,20,30,40,50,60,70,80,90 and the SD solid medium of 100mM boric acid on.26.5 cultivated 16 days under ℃, detect yeast and whether can on each substratum, form bacterium colony.
[screening of imparting boron-tolerance gene]
Use the expression library of Arabidopis thaliana (to provide by Nicolaus von doctor Wiren German, ホ one ヘ Application Ha イ system university; Schaaf G., Catoni E., Fits M., Schwacke R., Schneider A., von Wiren N., and Frommer W.B. (2002) A putative role for the vacuoler calcium/manganese proton antiporter AtCAX2 in heavy metaldetoxification.Plant Biol.4; 612-618.), by the Lithium Acetate method, transformed yeast 1169 strains.Transformed yeast is lined the SD substratum that has added 80mM boric acid, and (6.7g/l does not have amino acid whose yeast nitrogen base, 5g/l ammonium sulfate, 20g/l glucose, the 2g/l histidine, 2g/l methionine(Met), 3g/l leucine, 20g/l agar, pH5.5) on, cultivate down in 26.5 ℃.After 10 days-14 days, from the yeast that forms bacterium colony, reclaim plasmid.The plasmid that reclaims is imported yeast once more, confirm the reproducibility of boric acid resistance capacity.
[boric acid resistant proof]
Measure by spot test (spot assay) and liquid culture.Spot test (spot assay) is carried out according to the following steps.Vibration is cultivated each yeast until OD in the SD liquid nutrient medium 600Value reach 0.5-1.0.Be diluted to each zymic OD with the SD substratum 600Value identical.To OD 600Each yeast culture liquid of value unanimity, the diluent of preparation 1/5,1/25,1/125 and 1/625 dilution.With autospencer (Pipetman) (Gilson) with each diluent respectively bit by bit with 5 μ l point samples in SD solid medium that has added boric acid and the SD solid medium that does not add boric acid in contrast, with regard to same yeast, by the mode point sample of concentration attenuation from left to right.Point sample the zymic flat board cultivated about 10 days down at 30 ℃, observe the zymic growth and development state.
Mensuration by liquid culture is carried out as follows.In the SD liquid nutrient medium, vibrate under 30 ℃ and cultivate each yeast until OD 600Value reach about 1.0.Each nutrient solution is inoculated in continues in the SD liquid nutrient medium that contains boric acid and the not borated SD liquid nutrient medium in contrast to cultivate until OD 600Value be 0.1.30 ℃ of vibration cultivations down, per 24 hours mensuration OD 600Value.
[order-checking of imparting boron-tolerance gene]
The parsing of the base sequence that 6 cDNA that obtain by screening clone is carried out as follows.Carry out sequencing reaction by fluorescent dye terminator method, use ABI310 genetic analysis instrument to analyze base sequence.From the base sequence that obtains, determine the encoding gene of its base sequence by the BLAST retrieval of TAIR (http://www.arabidopsis.org/).
[The selection result of imparting boron-tolerance gene]
At first, the mensuration of the boric acid resistance capacity of yeast 1169 strains of using in carry out this experiment.With pYES2 and pYES2-BOR1 transformed yeast 1169.Use pYES2 and pYES2-BOR1 to transform to be because these carriers have the URA3 identical with the carrier FL61 that uses in the Arabidopis thaliana expression library that uses in the ensuing screening as selection markers.In addition, because the BOR1 expression of gene that the SD substratum is not induced pYES-BOR1 should be able to demonstrate the boric acid resistance that transforms same degree through pYES2.Will be through the yeast difference called after " 2 " and " 7 " of pYES2 and pYES2-BOR1 conversion." 2 " and " 7 " are cultivated in vibration in the SD liquid nutrient medium, line contain 0,20,30,40,50,60,70,80,90 and the SD solid medium of 100mM boric acid in.Its result shows, the yeast that transforms through above arbitrary carrier almost can not grow on the SD substratum that contains 80mM and the boric acid more than the 80mM (Fig. 1).
In order to separate the imparting boron-tolerance gene, by yeast 1169 strains are expressed, searching can contain the arabidopsis gene of growing on the SD substratum of 80mM in this experiment.So about 1,200,000 yeast that will transform through the expression library of Arabidopis thaliana line on the SD substratum that contains 80mM.Consequently, can obtain 80mM boric acid is demonstrated 46,66,72,84,86 and 87 these 6 transformed yeasts of resistance.The boric acid resistance of 46,72,84,86 and 87 the transformed yeast that obtains by spot test (spot assay) can be shown in Fig. 2 (for 66, as follows, because the coding gene identical with 46, so the result only represents 46).Yeast 1169 strains shown in the top of Fig. 2, contain on the SD substratum of 80mM and form bacterium colony hardly.On the other hand, these transformed yeasts can form more bacterium colony than arbitrary 1169 strains.Below, test by liquid culture.In the liquid culture, as shown in Figure 3, demonstrate than the about 3 times multiplication capacity of 1169 plant heights in the boric acid substratum in 46,72,86,87.But speed of growth difference is big in 84, with regard to the boric acid resistance, do not find with 1169 strains have the ability poor.In addition, when these genes being imported in the yeast BY4741 strain, equally also can give the boric acid resistance when importing to 1169 strains.The result of expression spot test (spot assay) among Fig. 4, the result that express liquid is cultivated among Fig. 5.When importing to the BY4741 strain, in all transformed yeasts, in liquid culture, also find there is ability (Fig. 5) in the boric acid resistance.
[order-checking of imparting boron-tolerance gene]
The base sequence that 6 cDNA that mensuration obtains through screening clone is retrieved the encoding gene of determining them by BLAST.Its result shows that 46 is consistent with AtPAB2 with 66, and 72 is consistent with AtMYB68, and 84 is consistent with AtMYB13, and is 86 consistent with AtRPS20B, 87 and the AtRBP47 unanimity.The sequence of each gene is shown in the following sequence table.AtPAB2 is the gene of coding polyadenylic acid conjugated protein, and AtMYB13 and AtMYB68 are the genes of coding MYB sample transcription factor, and AtRPS20 is the gene of coding ribosomal protein, and AtRBP47 is the gene of coding RNA conjugated protein.
Embodiment 2
[yeast strains and screening]
In this research, use yeast saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0), Y01169 (MATa his3D1 Leu2D0 met15D0 ura3D0 YNL275W ∷ kanMX4), Y04443 (MATa his3D1 leu2D0 met15D0 ura3D0 YGL076C ∷ kanMX4), and Y01094 (MATa his3D1 leu2D0 met15D0 ura3D0 YPL198W ∷ kanMX4).Can from BY4741, make up Y01169, Y04443, Y01094 strain (Winzeler et al., 1999) by insertional mutagenesis (insertional mutagenesis), obtain by EUROSCARF.
By Lithium Acetate method (Gietz and Schiestl, 1995), to clone in the Arabidopis thaliana cDNA library of expression plasmid pFL61 transformed yeast competent cell (Minet et al., 1992).Owing to compare, lacked discharge type boron vehicle promptly has susceptibility to boric acid YNL275W (following BOR1), so the Y01169 strain can be used as host's use (not display data) with the corresponding wild-type strain.Under 26.5 ℃, in the SD of the boric acid that contains 80mM solid medium (Sherman, 1991), screen transformant.The SD substratum contains varient necessary 2% glucose, 0.67% of growing up and does not contain amino acid whose yeast nitrogen base and 0.05% ammonium sulfate, the varient necessary amino acid (Met of the His of 20mg/L, the Leu of 30mg/L, 20mg/L) of growing, and with Tris pH is adjusted to 5.5.In order to form solid medium, add agar (2%w/v).By adding the boric acid of 80mM, the bacterium colony that can suppress non-conversion Y01169 (Δ bor1) cell fully forms.In the transformant, after cultivating for 2 weeks under 26.5 ℃, in containing the SD substratum of 80mM boric acid, select to form the cell of bacterium colony, in the presence of 80mM boric acid, check its situation of growing, confirm its resistance.For the phenotype of confirming to give by plasmid, separation quality grain, transformed yeast strain Y01169 again from positive isolate (positive isolate).The antagonism isolate carries out fluororotic acid and induces plasmid loss (fluoro-orotic acid-inducedplasmid loss) (Boeke, J.D., LaCroute, F. ， ﹠amp; Fink, G.R. (1984) Mol.Gen.Genet.197 345-346.), selects to demonstrate the clone of the dependent boric acid resistance of plasmid.
[structure of plasmid]
With the primer sets of table 1 record,, amplification AtRBP47c by PCR " the ORF sequence (canonical sequence number 34) of genes involved and RPL7B.With the subclone site (Promega society system) of extension amplification outcome to pGEM-T easy carrier.Handle these plasmids with NotI, will organize ORF fragment cloning (Minet et al, 1992) in the NotI site of expression vector pFL61 into resultant AtRBP45a, AtRBP47b, AtRBP47c, AtRBP47c ' and AtUBP1.In addition, with the ORF fragment cloning of RPL7B NotI site (Rentsch et al., 1995) to expression vector pDR195.PFL61 and pDR195 have PGK and the PMA1 promotor that is used to express respectively.
[table 1]
Gene ?Primer?sequences
AtRBP45a? ? AtRBP47a? ? AtRBP47b? ? AtRBP47c? ? AtRBP47c’? ? AtUBP1a? ? RPL7B? ? ?5’-AAAAAGCAGGCTTAATGCAGCAACCACCGTCAAACGCC-3’? 5’-AGAAAGCTGGGTTTCACTGACGTTGCTGCTGATAGTT-3’? 5’-AAAAAGCAGGCTTAATGCAGACACCAAACAACAACGGT-3’? 5’-AGAAAGCTGGGTTTCAAGAAGCTCCCGGGACTGCAGC-3’? 5’-AAAAAGCAGGCTTAATGCAGACAACCAACGGCTCAGAT-3’? 5’-AGAAAGCTGGCTTTCAATTCTCCCCATGATAGTTGTT-3’? 5’-AAAAAGCAGGCTTAATGGCAGACGTCAAGATTCAATCC-3’? 5’-AGAAAGCTGGGTTTCAGCTAACTTGTTGCTGATGACC-3’? 5’-AAAAAGCAGGCTTAATGGCAGACGTCAAGGTTCAATCC-3’? 5’-AGAAAGCTGGGTTTCAGCTAACTTGTTGCTGATGACC-3’? 5’-AAAAAGCAGGCTTAATGCAGAATCAAAGGCTTATTAA-3’? 5’-AGAAAGCTGGGTTTTACTGATAGTACATGAGCTGCTG-3’? 5’-AAAAAGCAGGCTTAATGTCCACTGAAAAAATCTT-3’? 5’-AGAAAGCTGGGTTTTAGTTCATAGCCTTAACCA-3’?
[analysis of boric acid resistance]
In order to analyze AtRBP47c " the boric acid resistance of relevant family gene, in yeast saccharomyces cerevisiae strain BY4741, import expression plasmid.In contrast, there is not the empty carrier of insertion to import among the BY4741 yet.Make transformant in the SD liquid nutrient medium, grow to stationary phase, the cell density of culture is adjusted to OD 600=1.0.The culture of having adjusted cell density is diluted to 1/5,1/25,1/125 and 1/625 with the SD liquid nutrient medium, the nutrient solution of dilution is dripped in the SD substratum of boric acid that 10 μ l contain or do not contain 80mM, cultivated 7 days down in 30 ℃.
In order to carry out the analysis in the nutrient solution, make transformant in the SD liquid nutrient medium, grow to stationary phase, the liquid nutrient medium that contains or do not contain 80mM boric acid with the SD liquid nutrient medium dilutes, and is adjusted to OD 600=1.0 to check the resistance capacity of high concentration of boric acid.
In order to analyze the boric acid resistance of Δ rpl7a (Y04443) varient and Δ rpl7b (Y01094) varient, use contains the SD substratum that 2% glucose, 0.67% amino acid do not contain the ammonium sulfate of yeast nitrogen base and 0.05%, with Tris pH is adjusted to 5.5, adds essential amino acid (Met of the His of 20mg/L, the Leu of 30mg/L, 20mg/L and the Ura of 20mg/L) again.Obtain varient from EUROSCARF.In order further to investigate the effect of RPL7B in the boric acid resistance, make RPL7B superfluous expression in yeast strains Y04443.Carry out the analysis of boric acid resistance by the way.
[detecting non-montage transcript] by RT-PCR
Yeast cell grows to logarithmic phase (OD in the SD liquid nutrient medium 600=0.5～1.0), the final concentration with 80mM adds boric acid.30 ℃ cultivate down after 24 hours, take out the 1ml sample, reclaim under cell, the liquid nitrogen by centrifugation freezing, in-80 ℃ of preservations until use.
Use RNeasy Mini Kit (Qiagen corporate system) from yeast cell, to extract total RNA, use MuLV reversed transcriptive enzyme (Applied Biosystem society system) and olig (dT) 16Total RNA of primer reverse transcription 1 μ g.To 1/15 of RT product, with following circulation carry out PCR:94 ℃ following 30 seconds, 45 ℃ following 30 seconds, 72 ℃ following 1 minute, carry out 40～50 times.In Smart Cycler (Cepheid society system), use archaeal dna polymerase ExTaq (Takara society system) to carry out PCR.The primer sets of using in this analysis is recorded in the table 2, is delivered as the side information on the webpage of PNAS.The transcript that separates amplification on 2% sepharose detects after the dyeing of Etd bromine.
[table 2]
Gene Primer?sequences
SNR17A? ? SNR17B? ? YBR230C? ? VMA10? ? SEC27? ? YNL050C? ? RPL7B? ? 5’-AATCTGTGTCGACGTACTTC-3’(Forward)? 5’-AGAAGTACATAGGATGGGTC-3’(Reverse)? 5’-AAAAATTGTCGACGTACTTC-3’(Forward)? 5’-AAAGGAAGTTATCACAATTG-3’(Reverse)? 5’-CCAGCATCTATGTCTGCAAC-3’(Forward)? 5’-CGTATCTGGAGTAGTATTTC-3’(Reverse)? 5’-GCAAGGTATACAAAGCAGAA-3’(Forward)? 5’-TCATCCTTTTTCTTCTCTGC-3’(Reverse)? 5’-GACACGATGAAGTTGGATAT-3’(Forward)? 5’-TGACTGTCAAATCATCACTG-3’(Reverse)? 5’-CAGTATAAAAATGTCTGAAT-3’(Forward)? 5’-TGGTTGATTATTTCTTCTTC-3’(Reverse)? 5’-ATCAACGTCATAATGTCCAC-3’(Forward)? 5’-TACCAGAGTTGATTCTTGTC-3’(Reverse)?
MUD1? ? SNC1? ? POP8? ? ARP2? ? CNB1? ? RPS22B? ? YML025C? ? TUB3? ? STO1? ? RPS16A? ? SAR1? ? PMI40? ? RPL7A? ? YBL091C-A? ? RPL19A? ? PCH2? ? 5’-ACCTAAAGAAACCATGTCAG-3’(Forward)? 5’-TATCAAGGTTGTACGTTTCG-3’(Reverse)? 5’-ATGTACAGTCTAAGTCAAGG-3’(Forward)? 5’-GACTAAAGTGAACAGCAATG-3’(Reverse)? 5’-GAGAATGGCAATATTTCAAG-3’(Forward)? 5’-TGTTCTTCTTCTTCCATTAC-3’(Reverse)? 5’-TGGACCCACATAATCCAATT-3’(Forward)? 5’-TTTCGAACATTACCTCACAC-3’(Reverse)? 5’-GTGGATGGTCTTTTAGAAGA-3’(Forward)? 5’-AACTCCTCGAAACTTAAACG-3’(Reverse)? 5’-TATTGAGACCTTCTTCCAAG-3’(Forward)? 5’-AAGATTTTACCGGAAACGTG-3’(Reverse)? 5’-GACGATAAAAAGAAATTTGGTG-3’(Forward)? 5’-CTCAAAGCGTTGTTGAAAG-3’(Reverse)? 5’-GAGAGAGGTCATTAGTATTA-3’(Forward)? 5’-TTTTCTAATAACAGGGAACC-3’(Reverse)? 5’-GTTTAATAGAAAAAGAAGAGGAG-3’(Forward)? 5’-TAGTTCATCAACTAAAAACATGG-3’(Reverse)? 5’-AGCTGTCCCAAGTGTTCAA-3’(Forward)? 5’-ACCCTTACCACCGAATTTC-3’(Reverse)? 5’-GTTGGGATATTTTTGGTTGG-3’(Forward)? 5’-AAAGGAACGTCCTTCAATTC-3’(Reverse)? 5’-AACAAGCTGTTCAGGTTAGA-3’(Forward)? 5’-GGTTTGTGATTATCATCAGG-3’(Reverse)? 5’-AATTAAAGATCACAATGGCCG-3’(Forward)? 5’-CTTGGTAACTTTGACGAATG-3’(Reverse)? 5’-CAGAAAAGCTGGTGTTCAAG-3’(Forward)? 5’-TGATTCTGCATCGTGGTTTC-3’(Reverse)? 5’-TTGATTAAGAACTCCAAAGC-3’(Forward)? 5’-TCTTCTCAAGACACGTAATC-3’(Reverse)? 5’-AGATGAGGTTGAAGCAATAG-3’(Forward)? 5’-CAAGGGCAATTTCCTTATTG-3’(Reverse)?
RPS9B? ? YBR230C? ? YDR381C-A? ? YRA1? ? UBC8? ? MND1? ? MER3? ? ERV1? ? SRB2? ? MOB1? ? RPS21A? ? NYVI? ? YLR211C? ? TAD3? ? ERV41? ? SPO1? ? 5’-TAAGACTAAGCAACAATGCC-3’(Forward)? 5’-AAACCCAACTTGTAGACTTG-3’(Reverse)? 5’-GCATCTCATAATATGTCTGC-3’(Forward)? 5’-TTGTTGCTAAGACTGTAGAG-3’(Reverse)? 5’-CAAATCCATTTCAAAATATAGG-3’(Forward)? 5’-CTCCTCCTATCTAAAAAACC-3’(Reverse)? 5’-AAGAAGAGTTGGTAAGCAAG-3’(Forward)? 5’-CACCGTTTTTGAATGTGATG-3’(Reverse)? 5’-AGCGTAATACGAAAGATGAG-3’(Forward)? 5’-AGCTTCGTTATTCAAGGGAT-3’(Reverse)? 5’-GTATCATAAACATTCAACAATG-3’(Forward)? 5’-CGGATCTGTTGTTTATTCTC-3’(Reverse)? 5’-AAACAAAGTTTGATCGCCTC-3’(Forward)? 5’-TCGTGCTCAAACATTTCTTC-3’(Reverse)? 5’-AAAATGACGGATAATCCACC-3’(Forward)? 5’-TTCAAAGTCTTTAGCACACC-3’(Reverse)? 5’-CAATCCATCATGGGAAAATC-3’(Forward)? 5’-CTTGGACGACAAAATAGTGT-3’(Reverse)? 5’-AGGACTTCAATTTCCATGTC-3’(Forward)? 5’-AGTGTCATCTCCACAATTTG-3’(Reverse)? 5’-GAAAACGATAAGGGCCAATT-3’(Forward)? 5’-CGTTCTTTAACAAACCATCG-3’(Reverse)? 5’-TACCAAATGAAACGCTTTAATG-3’(Forward)? 5’-TCTTCATGGAAAGAGTCTAG-3’(Reverse)? 5’-ATGGAATGAGTACTTTAGCG-3’(Forward)? 5’-CTTCATTTCCGAGTTTTTGG-3’(Reverse)? 5’-AATAGAAAATCGGCTTCTGC-3’(Forward)? 5’-TATTTGATCATTGGGGTTGC-3’(Reverse)? 5’-GATTGAAGACATTTGATGCG-3’(Forward)? 5’-TCGCCACTAACTCTATTTAC-3’(Reverse)? 5’-ACCATTTCAGGTACAATGTC-3’(Forward)? 5’-CTTCGGAAATATCGAATTCC-3’(Reverse)?
YOL048C? ? RPS9A? ? CIN2? ? YPR098C? ? 5’-CTGAAACGATACCAACAATG-3’(Forward)? 5’-TTTGTGGTTTAGGCAATACC-3’(Reverse)? 5’-ATACAAAAGTATACAACATGCC-3’(Forward)? 5’-TTTCCAAGAAATCTTCGACC-3’(Reverse)? 5’-CTTTACTGCGAAGATAAAGG-3’(Forward)? 5’-GCCACTATAATCTGTTGTTG-3’(Reverse)? 5’-TCAAAACTACGGCTCATTTG-3’(Forward)? 5’-TGAACAAAAGACTCAATCCG-3’(Reverse)?
[analysis of salt resistance]
Analyze for the salt resistance, except use contained the SD substratum of NaCl of 1.75M or 2M, all the other were undertaken by above-mentioned boric acid resistance analysis.
[accession number]
The sequence GenBank number of landing of record is as follows among the embodiment 2.Arabidopsis thaliana sequence AtRBP45a, MN124872; AtRBP45b, MN101037; AtRBP45c, MN118834; AtRBP45d, MN121940; AtRBP47a, MN103848; AtRBP47b, MN112800; AtRBP47c, MN103642; AtRBP47c ', MN103643; AtUBP1a, MN104285; AtUBP1b, MN101598; AtUBP1c, MN112266; And yeast saccharomyces cerevisiae sequence RPL7A, X62627; RPL7B, Z7355.
[giving the Arabidopis thaliana cDNA clone's of high concentration of boric acid resistance separating resulting in the yeast]
With Arabidopis thaliana cDNA expression library (Minet, M., Dufour, M.-E. ， ﹠amp; Lacroute, F. (1992) Plant J.2, these transformant are selected in 417-422.) transformed saccharomyces cerevisiae strain Y01169 strain in the ware of the boric acid that contains 80mM.The boric acid of this concentration, the bacterium colony that also can suppress the Y01169 cell after cultivating for 2 weeks under 26.5 ℃ fully forms.By this screening, isolate several boric acid resistance enhanced zymic bacterium colonies that demonstrate.1 among the cDNA clone demonstrates coding RNA conjugated protein AtRBP47c '.
[AtRBP47c ' Expression of Related Genes in Arabidopis thaliana source gives yeast boric acid resistance]
AtRBP47c ' has 3 RNA identification motifs (RRM).The proteinic gene that has 11 kinds of codings to have 3 RRM and have the sequence identity score more than 100 for AtRBP47c ' in the arabidopsis gene group through the calculating of BLASTP program.The dendrogram of the relevant family protein of these AtRBP47c ' is shown in Fig. 6 A.
For whether the expression of investigating these arabidopsis genes gives yeast boric acid resistance, the clone is corresponding to 6 kinds of gene ORF sequences (AtRBP45a, AtRBP47a, AtRBP47b, AtRBP47c, AtRBP47c ' and AtUBP1a) in expression vector pFL61.In yeast strains BY4741, import plasmid, investigate the boric acid resistance of these transformant.Shown in Fig. 6 B, 6 kinds of constructs all give yeast post energy for growth in containing the SD substratum of 80mM boric acid in various scopes.In order to compare the boric acid resistance level of these transformant, in liquid culture, analyze the growth rate in the presence of the boric acid.All transformant are all compared according to more early showing growth rate.Among the figure, shown that the transformant of expression AtRBP47c ' shows growth rate (Fig. 6 C) the earliest.
[boric acid is handled the montage that suppresses RPL7B in the yeast, but does not suppress the montage of RPL7A]
The inventor finds that the surplus expression of AtRBP47c ' genes involved gives the boric acid resistance.The effect of these genes of Arabidopis thaliana is not clear, but other floristic similar gene can give feature.Tobacco Whiteflower Leadword Root (Nicotianaplumbaginifolia) RBP45 (Simpson, C.G., Jennings, S.N., Clark, G.P., Thow, G. ， ﹠amp; Brown, J.W.S. (2004) Plant J.37,82-91) and UBP1 (Lambermon, M.H., Simpson, G.G., Wieczorek Kirk, D.A., Hemmings-Mieszczak, M., Klahre, U. ， ﹠amp; J.19, Filipowicz, W. (2000) EMBO 1638-1649) demonstrate montage efficient and strengthen.So, in 20 kinds by the optional yeast saccharomyces cerevisiae of the RT-PCR genes that contain intron, investigation boric acid is to the influence of montage.In 6317 kinds of nuclear genes in the genes of brewing yeast group only 231 kinds of genes contain intron (Munich Information Center for Protein Sequences:http: //mips.gsf.de/genre/proj/yeast).In investigated 20 kinds of genes, relatively Ji Lei non-montage fragment and montage fragment must be observed the segmental increase of non-montage of handling generation through boric acid by the proteinic gene RPL7B of ribosomal large subunit by encoding.The montage of this prompting RPL7B in the yeast that boric acid is handled is suppressed (Fig. 7 B).
RPL7B contains 2 introns.The segmental size of non-montage demonstrates these fragments through the montage of the 1st or the 2nd intron and (Fig. 7 A with reference to).In order to study which intron boric acid is had more susceptibility, clone non-montage fragment, measure 8 clones' dna sequence dna.6 and 2 s' clone contains the 1st and the 2nd intron respectively.This prompting all suppress among both at the 1st and the 2nd intron, but the 1st intron has more susceptibility to high concentration of boric acid.These results also represent, 1 correct montage in 2 introns that is to say that these non-montage fragments are not that genomic dna forms because of pollution, are that the reverse transcription reaction by RNA produces.
And then, in the yeast of expressing AtRBP47c ', do not observe the montage that produces by boric acid and suppress (Fig. 7 B) RPL7B.This results suggest, AtRBP47c ' rises the montage efficient of RPL7B in the presence of high concentration of boric acid.The enhancing of montage efficient may be the reason of yeast mesoboric acid resistance.
RPL7B has homologous (バ ラ ロ ゲ) RPL7A (sequence number 32) in the yeast genes group.RPL7A gene (sequence number 31) has 2 introns as the RPL7B gene.Investigated the influence of boric acid to the montage of RPL7A.Different with the situation of RPL7B, do not observe the montage inhibition (Fig. 7 C) that boric acid produces.
[because the destruction of RPL7A is reduced the boric acid resistance in the yeast]
The dual destructive varient of RPL7A and RPL7B be lethality (yeast genes group database (SaccharomycesGenome Database): Http:// db.yeastgenome.org), demonstrating RPL7 protein is that yeast growth is grown necessary.If consider the difference of 2 gene mesoboric acid susceptibility, just exist RPL7A to destroy the discrepant possibility of boric acid resistance of varient and RPL7B destruction varient for montage.Δ rpl7b (Y01094) demonstrates the boric acid resistance level equal with the wild-type yeast saccharomyces cerevisiae, but the boric acid resistance of Δ rpl7a (Y04443) is than wild-type lower (Fig. 8 A).Even at the difference of liquid culture mesoboric acid resistance also clearly (Fig. 8 B).The RPL7B montage inhibition that these results suggest are produced by boric acid is because the boric acid resistance minimizing of Δ rpl7a causes.
[RPL7A destroys in the yeast because the rising of the boric acid resistance that expression produced of intronless RPL7B]
The minimizing of the boric acid resistance of Δ rpl7a is if because the minimizing of the RPL7 protein level that suppress to be produced by the RPL7B montage forms, and the expression of intronless RPL7B cDNA makes the resistance rising of Δ rpl7a really.
Whether the expression of intronless RPL7B makes the boric acid resistance rise among the investigation Δ rpl7a.With the ORF sequence clone of RPL7B in expression vector pDR195.Then, plasmid is imported Δ rpl7a, the boric acid resistance of investigation transformant.Shown in Fig. 8 C, the expression of intronless RPL7B is risen the boric acid resistance of Δ rpl7a.Its result shows that the inhibition of RPL7B montage is the reason that is stopped by the growth that the high concentration of boric acid of Δ rpl7a produces.
[handling the analytical results that montage suppresses in the gene that contains atypical tapping point sequence (noncanonical branchpoint sequnce) that produces by boric acid]
RPL7B has atypical tapping point sequence (table 3 reference) in the 1st intron.231 kinds of nuclears contain in the gene of intron, have found 28 kinds of genes that contain this atypical tapping point sequence.In these 28 kinds of genes, observe with the montage fragment in 9 kinds of genes and compare, handle the segmental level rising of the non-montage that produces (Fig. 9) through boric acid.These genes are ERV1, ERV41, NYV1, RPS9A, RPS9B, SRB2, YOL048C, YPR098C and YRA1.
[table 3]
Expression in the table 3,3 consensus sequences, 5 ' splice site, tapping point and the 3 ' splice site confirmed in the yeast.Show with the white with black word table in the tapping point of the 1st intron by the transformation point of A to G.Y refers to pyrimidine ribonucleotide (C or U).
The surplus of analysis AtRBP47c ' is expressed the influence to the montage inhibition of these genes that produced by boric acid.As shown in Figure 9, in the yeast of expressing AtRBP47c ', the montage inhibition level of NYV1 and SRB2 is hindered.NYV1 and SRB2 encode respectively v-SNARE protein and rna plymerase ii holoenzyme protein.It is the enhancing of montage efficient that these results point out the surplus of AtRBP47c ' to express the mechanism that gives yeast boric acid resistance forcefully.
[it is different with the influence of being handled generation by boric acid that salt is handled the influence that produces]
The surplus of splicing factor gene express give yeast and/or plant salt resistance (Forment, J., Naranjo, M.A., Roldan, M., Serrano, R.; Vicente, O. (2002) Plant J.30,511-519., 2002; Serrano, R., Gaxiola, R., Rios, G., Forment, J., Vicente, O. ， ﹠amp; Ros, R. (2003) Monatsh.Chem.134,1445-1464.).Also investigate AtRBP47c ' genes involved and whether give yeast salt resistance.For 6 kinds of AtRBP47c ' genes involveds checking in this research, the salt resistance is all less than rise (Figure 10 A) in the yeast.And then, be exposed to the montage of not observing RPL7B in the cell under the salt (high salt) of high density and suppress (Figure 10 B).These results seemingly AtRBP47c ' genes involved do not have function for the salt resistance, and it is unique that the inhibition of RPL7B montage is handled for boric acid.
[investigation]
Intact by zymic complementary action (Fill) from Arabidopis thaliana, separate AtRBP47c ' as the gene that gives yeast cell boric acid resistance.In the yeast genes group, the proteinic gene that has 7 kinds of codings to have 3 RRM and have the sequence identity score more than 100 for AtRBP47c ' through the calculating of BLASTP program.In these the gene, with the similar gene of AtRBP47c ' be NAM8.NAM8 is inhibitor separated (Ekwall, K., Kermorgant, M., Dujardin, G., Groudinsky, the O. ， ﹠amp as the montage shortage of original grain line body; Slonimski, P.P. (1992) Mol.Gene.Genet.233 136-144.), by analysis thereafter, demonstrates NAM8 and Ulsn RNA and interacts, when this processing was suppressed owing to the existence that has atypical 5 ' splice site, NAM8 was necessary (Gottschalk, A., Tang, J. of effective identification of 5 ' splice site, Puig, O., Salgado, J., Neubauer, G., Colot, H.V., Mann, M., Seraphin, B., Rosbash, M., Luhrmann, R. ， ﹠amp; Fabrizio, P. (1998) RNA 4,374-393.; Puig, O., Gottschalk, A., Fabrizio, P. ， ﹠amp; Seraphin, B. (1999) Gene.Dev.13,569-580.).According to these observationss, suppose that AtRBP47c ' exercises same function with NAM8 aspect the boric acid resistance.But, the surplus of NAM8 is expressed and is not given yeast boric acid resistance, NAM8 destruction varient and wild-type are same, demonstrate the boric acid resistance, and AAtRBP47c ' demonstrates and other stage of montage processing and/or the relevant possibility of other reaction (1 or a plurality of) of boric acid resistance.
In this research, in yeast, in optional 20 kinds of genes, find that boric acid can suppress the montage (Fig. 7 B) of RPL7B.The dna sequence analysis of the 1st intron by analyzing this gene has been known that the 1st intron exists at the consensus sequence of tapping point to transform.As shown in table 1, the 2nd A is converted into the G in the 1st intron of RPL7B in the tapping point consensus sequence.(combining of the tapping point of Branchpoint bridging protein (BBP) is that middle important step (Abovich and Rosbash, 1997) is carried out in montage with the tapping point cross-linked proteins.Affinity between present known BBP and tapping point sequence is the important factor (Champion-Arnaud, et al., 1995) of montage efficient.Especially, it is reported that A in the 2nd Nucleotide of this tapping point sequence makes with the BBP affinity to the conversion of G reduces about 10% (Berglund, J.A., Chua, K., Abovich, N., Reed, R. ， ﹠amp; Rosbash, M. (1997) Cell 89,781-787.).So, one of gene that RPL7B montage efficient seemingly is lower.
It is reported, in the Hela cells in vitro splicing system, the 2nd step of montage handles and be suppressed (Shomron, N. ， ﹠amp owing to boric acid; Ast, G. (2003) FEBS Lett.552,219-224.).The 2nd step of montage is the processing of 3 ' terminal ligation of processed exon being arrived 5 ' end of next exon.If consider that boric acid combines (Ralston, N.V.C. ， ﹠amp by ribose with cis-diol; Hunt, C.D. (2000) FASEB J.14, A538.; Nicholas et al., 2001; Ricardo, A., Carrigan, M.A., Olcott, A.N. ， ﹠amp; Benner, S.A. (2004) Science303,196.), as if the ligation reaction in the 2nd step of montage owing to be suppressed with 3 ' combining of end of the exon of boric acid.According to above-mentioned Shomron and Ast (2003),, think that it is general phenomenon that the montage that is produced by boric acid in the 2nd step suppresses because 5 kinds of different mRNA precursor substances demonstrate same inhibition.Montage inhibition in the yeast takes place in this situation, all introns really equally.But in 20 kinds of genes checking in the initial step of this research, the gene of observing inhibition only is RPL7B (Fig. 7 B).
This specific specificity suppresses and can illustrate as follows.In the 2nd step zymic all contain the montage that all takes place in the gene of intron to produce and suppress by boric acid.In the time of should reducing the montage medium that contains intron at once in this step, when carrying out montage usually, contain the montage medium accumulation of intron.The accumulation of medium suppresses conventional metabolic turnover.The gene that this situation seemingly has the inefficient intron of montage is strong for the susceptibility that the montage that is produced by boric acid suppresses.1 example as this gene can exemplify the gene that contains atypical tapping point sequence as RPL7B.To have other gene of same characteristic features, suppress by analyzing the montage that produces by high concentration of boric acid, check this supposition (Fig. 9).In this analysis, find that the high concentration of boric acid processing suppresses montage in the 9 kinds of genes that contain atypical tapping point sequence beyond the RPL7B.This result shows that clearly a kind of the toxic mechanism of boric acid is that the montage with gene of the inefficient intron of montage suppresses.And then, in the gene of these 9 kinds, find that the montage of 2 kinds of genes suppresses to hinder (Fig. 9) because the surplus expression of AtRBP47c ' is suppressed.Perhaps, it is that the enhancing of montage efficient of a part of gene of a plurality of genes of being suppressed of montage realizes in handling by high concentration of boric acid that this results suggest is expressed the boric acid resistance that produces by the surplus of AtRBP47c '.Therefore, perhaps the montage of the gene of restricted number inhibition is growth inhibiting reason.
AtRBP47c ' related protein has 3 RRM.The RNA of UBP1 that has confirmed RBP45, RBP47 and N.plumbaginifolia is in conjunction with activity.These protein all has and U-rich sequence bonded tendency (Lambermon, M.H., Simpson, G.G., Wieczorek Kirk, D.A., Hemmings-Mieszczak, M., Klahre, U. ， ﹠amp; Filipowicz, W. (2000) EMBO J.19,1638-1649.:Lorkovi?, Z.J., Wieczorek Kirk, D.A., Klahre, U., Hemmings-Mieszczak, M. ， ﹠amp; Filipowicz, W. (2000) .RNA 6,1610-1624.).And then, need at least 2 RRM (Lorkovic et al., 2000) in the deletion analysis demonstration of the RBP45 of N.plumbaginifolia and the interaction of RNA.Recognize gradually that now RRM is relevant with the RNA combination, but certain proteinic RRM participates in and other protein interactions (Kielkopf, C.L., Lucke, S. ， ﹠amp; Green, M.R. (2004) Gene Dev.18,1513-1526.).Especially, it is reported the splicing factor yeast U2AF that contains 3 RRM 65The 3rd RRM and BBP (Rain, J.C., Rafi, Z., Rhani, Z., Legrain, P. ， ﹠amp; Kramer, A. (1998) RNA 4,551-565.).These results suggest AtRBP47c ' also with the interactional possibility of BBP.And then there is the U-rich sequence in 3 ' end of the 1st intron of RBP7B and the analysis revealed tapping point of SRB2 intron sequences.If comprehensively these results suppose that AtRBP47c ' by combining with BBP, stablizes the interaction of BBP and tapping point, stablize the U-rich sequence of tapping point.Consequently, montage efficient increases.
It is reported that montage is owing to salt pressure is suppressed.And then it is reported that the surplus of a plurality of splicing factors of SR protein etc. is produced increases salt resistance (Forment, J., Naranjo, M.A., Roldan, M., Serrano, R. ， ﹠amp in yeast and the plant; Vicente, O. (2002) Plant J.30,511-519.., 2002; Serrano, R., Gaxiola, R., Rios, G., Forment, J., Vicente, O. ， ﹠amp; Ros, R. (2003) Monatsh.Chem.134,1445-1464.).In this research, the surplus of AtRBP47c ' genes involved is expressed and is not given yeast salt resistance (Figure 10 A), and the montage that does not detect RPL7B after salt is handled suppresses (Figure 10 B).These results suggest salt handle and boric acid handle in montage inhibition machine-processed different.
Embodiment 2 shows that the key of the toxic mechanism of boric acid is the specificity inhibition of montage, and the gene relevant with the enhancing of montage efficient brings the report the earliest of boric acid resistance.But, have the toxic mechanism beyond the inhibition of montage really.Reason is, even also observe the toxic influence of boric acid in the prokaryotic organism that do not carry out montage.
Description of drawings
Fig. 1 is the result's of the expression boric acid resistance capacity of using yeast 1169 strains figure.With pYES2 " 2 " and 1169 strains of pYES2-BORI " 7 " transformed yeast.Each yeast is lined respectively on the SD substratum of the boric acid that contains 0-100mM.26.5 ℃ of results that cultivate 16 days have down been shown.
Fig. 2 is result's the figure that is illustrated in the growth of yeast 1169 strains in the superfluous substratum of boric acid.With 46,72,84,86 and 8 transform 1169 strains of 7 yeast.Behind each yeast of liquid culture, line on the SD substratum of the boric acid that contains 80mM.The site slowly dilutes 1/5 from left to right.26.5 ℃ of results that cultivate 9 days have down been represented.
Fig. 3 is result's the figure of the boric acid resistant proof of yeast 1169 strains in the express liquid substratum.
With 46,72,1169 strains of 84,86 and 87 transformed yeasts.Behind each yeast of liquid culture, be inoculated in and continue in the SD substratum of the boric acid that contains 80mM to cultivate until OD 600Value be 0.1.Cultivated 4 days down, measure OD for 30 ℃ 600Value.Carry out 3 experiments.Represent with figure with the standard deviation of mean value.
Fig. 4 is the figure of the growth result of the yeast BY4741 strain in the superfluous substratum of expression boric acid.
With 46,72,84,86 and 87 transformed yeast BY4741 strains.Behind each yeast of liquid culture, point sample is in the SD of the boric acid that contains 100mM substratum.The site slowly dilutes 1/5 from left to right.26.5 ℃ of results that cultivate 10 days have down been represented.
Fig. 5 is result's the figure of the boric acid resistant proof of the yeast BY4741 strain in the express liquid substratum.
With 46,72,84,86 and 87 transformed yeast BY4741 strains.After each yeast liquid culture, be inoculated in and continue in the SD substratum of the boric acid that contains 80mM to cultivate until OD 600Value be 0.1.Cultivated 4 days down, measure OD for 30 ℃ 600Value.Carry out 3 experiments.Represent with figure with the standard deviation of mean value.
Fig. 6 is result's the figure of the boric acid resistance of expression AtRBP47c ' genes involved transformed yeast cell.
(A) dendrogram of the relevant family gene of AtRBP47c '.Dendrogram is represented the relative evolutionary distance of the relevant family protein of AtRBP47c ', adopts the NJ method to make.Bar is represented the gene distance at 0.1 aminoacid replacement/position.
(B) solid medium mesoboric acid resistance.Yeast cell growth is to OD 600Be 1.0, serial dilution, then splash into 10 μ l added 0 or the SD ware of the boric acid of 80mM in.Cultivated back record growth in 10 days.Use through empty carrier pFL61 transformed yeast cells in contrast.
(C) liquid nutrient medium mesoboric acid resistance.Yeast cell growth is to OD 600Be 1.0, added 0 or the SD substratum of the boric acid of 80mM in be diluted to OD 600Be 0.1.30 ℃ of yeast cell of cultivating dilution down, the record hour OD after the record dilution 600Value.Horizontal stripe is represented the standard deviation of the mean value ± mean value of 3 mensuration.
Fig. 7 is the figure of the influence of expression montage mesoboric acid.
(A) sketch map of the montage of RPL7B.By montage can be from the pre-mRNA of RPL7B 3 kinds of mRNA of output.Arrow refers to the position of the primer that uses among the RT-PCR.
(B) boric acid is to the influence of the montage of RPL7B.Yeast cell growth is to OD 600Be 1.0, with the final concentration interpolation boric acid of 80mM.After 24 hours, reclaim yeast cell, separate total RNA.Synthetic cDNA from total RNA is as carry out the template that montage is analyzed by PCR.During this is analyzed, use the yeast strains BY4741 (wild-type) that transforms through empty carrier pFL61 or AtRBP47c ' expression vector (AtRBP47c ').
(C) boric acid is to the influence of the montage of RPL7A.In the BY4741 that transforms through PFL61, analyze the montage of RPL7A by RT-PCR.
Fig. 8 is the result's of expression RPL7A or the RPL7B boric acid resistance of destroying yeast cell figure.
(A) solid medium mesoboric acid resistance.Yeast cell growth is to OD 600Be 1.0, serial dilution, then splash into 10 μ l added 0 or the SD ware of the boric acid of 80mM in.Cultivated back record growth in 7 days.
(B) liquid nutrient medium mesoboric acid resistance.Yeast cell growth is to OD 600Be 1.0, added 0 or the SD substratum of the boric acid of 80mM in be diluted to OD 600Be 0.1.Dilute back 30 ℃ of yeast cell 21 hours (SD) and 60 hours (boric acid of SD+80mM) of cultivating dilution down, write down OD then 600Value.Horizontal stripe is represented the standard deviation of the mean value ± mean value of 3 mensuration.Δ rpl7a and Δ rpl7b represent that respectively RPL7A destroys varient (Y0443) and RPL7B destroys varient (Y01094).
(C) surplus of RPL7B is expressed the influence that RPL7A is destroyed yeast mesoboric acid resistance.Yeast cell growth is 1.0 to OD600, serial dilution, then splash into 10 μ l added 0 or the SD ware of the boric acid of 80mM in.Cultivated back record growth in 5 days.Use through empty carrier pFL61 transformed yeast cells in contrast.
Fig. 9 is the figure of expression boric acid to the influence of the montage of the gene that contains atypical tapping point sequence.
Yeast cell growth is to OD 600Be 1.0, then the final concentration with 80mM adds boric acid.After 24 hours, reclaim yeast cell, separate total RNA, as carry out the template that montage is analyzed by PCR.During this is analyzed, use the yeast strains BY4741 (wild-type) that transforms through empty carrier pFL61 or AtRBP47c ' expression vector (AtRBP47c ').What white and black arrow pointed to respectively is non-montage fragment, montage fragment.
Figure 10 is the growth of expression salt pair AtRBP47c ' genes involved transformed yeast cell and to the figure of the influence of the montage of RPL7B.
(A) salt resistance in the solid medium.Yeast cell growth is to OD 600Be 1.0, serial dilution then splashes at 10 μ l and has added 0,1.75 or the NaCl of 2M.Cultivate record growth after 7 days.Use through empty carrier pFL61 transformed yeast cells in contrast.
(B) influence of the montage of salt pair RPL7B.Yeast cell growth is to OD 600Be 1.0, add NaCl or boric acid with the final concentration that is respectively 2M or 80mM.After 24 hours, reclaim yeast cell, separate total RNA.Synthetic cDNA from total RNA is as carry out the template that montage is analyzed by PCR.
Sequence table
＜110〉independent administrative legal person's science and technology development organization (Japan Science and Technology Agency
＜120〉proteins imparting boron-tolerance and gene thereof
<130>K05327PCT
<150>JP2004-073324
<151>2004-03-15
<160>134
<170>PatentIn?version?3.1
<210>1
<211>1332
<212>DNA
＜213〉Arabidopis thaliana
<400>1
atgttgctca?atgacaagca?agtgtatgtg?ggtcctttcc?tgaggagaca?agaaagagac 60
tccactgcta?acaaaacgaa?attcaccaat?gtgtatgtga?agaatctcgc?ggaaagtact 120
accgatgatg?acttgaagaa?tgcttttggc?gagtatggaa?agataacaag?tgctgtcgtg 180
atgaaagatg?gagaagggaa?gtccaagggc?tttgggtttg?tcaactttga?aaatgctgat 240
gatgctgcta?gggctgtgga?gtctctcaat?gggcacaaat?ttgatgataa?ggagtggtat 300
gttggtagag?cccagaagaa?gtcagagagg?gaaacagaat?taagggtccg?ttatgaacag 360
aatttgaagg?aagctgcaga?caagtttcaa?agttcaaact?tgtatgttaa?gaatttggat 420
cctagcattt?cagatgagaa?acttaaagag?atcttttctc?cttttggtac?cgttacatct 480
agcaaggtga?tgcgggatcc?taatggaaca?agcaaaggct?caggttttgt?tgctttcgca 540
actcccgaag?aagcaactga?agctatgtca?cagttgagcg?gtaaaatgat?cgaaagcaag 600
ccactctatg?tggctattgc?acagcggaag?gaagacagaa?gggtcagact?acaggctcag 660
ttttcccaag?tgaggccagt?tgcaatgcag?ccgtctgttg?gtccccgcat?gccagtgtat 720
cccccgggtg?gtcctggtat?tggacaacaa?atgttctatg?gtcaggcccc?tcctgccatg 780
attcctcccc?agcctgggta?tggataccaa?cagcagcttg?ttcctggaat?gagacctggt 840
gggggtcctg?tacccagttt?cttcatgcct?atggttcagc?cacagcagca?gcgtcctgga 900
ggaggaagac?gtcctggggg?aatccaacac?tcccagcagc?aaaatcccat?gatgcagcaa 960
cagatgcatc?caaggggtcg?gatgttccgg?tatccccaag?ggcgtggtgg?tagtggtgat 1020
gtgcctccat?atgatatggg?caacaacatg?ccattgacta?ttggagcttt?ggcttcaaat 1080
ctgtctaatg?ctactccaga?gcaacagagg?acgatgctgg?gtgaggtgct?gtacccgttg 1140
gtggagcagg?ttgaggcaga?gtctgcagcc?aaagtgactg?ggatgctttt?ggagatggac 1200
cagactgaag?tgctccatct?gttggagtca?ccagaagctc?tcaaggccaa?agttgcagag 1260
gctatggatg?ttctcaggag?tgtcgctgct?ggtggtgcaa?ccgagcagct?cgcttccttg 1320
aacctctctt?aa 1332
<210>2
<211>443
<212>PRT
＜213〉Arabidopis thaliana
<400>2
Met?Leu?Leu?Asn?Asp?Lys?Gln?Val?Tyr?Val?Gly?Pro?Phe?Leu?Arg?Arg
1 5 10 15
Gln?Glu?Arg?Asp?Ser?Thr?Ala?Asn?Lys?Thr?Lys?Phe?Thr?Asn?Val?Tyr
20 25 30
Val?Lys?Asn?Leu?Ala?Glu?Ser?Thr?Thr?Asp?Asp?Asp?Leu?Lys?Asn?Ala
35 40 45
Phe?Gly?Glu?Tyr?Gly?Lys?Ile?Thr?Ser?Ala?Val?Val?Met?Lys?Asp?Gly
50 55 60
Glu?Gly?Lys?Ser?Lys?Gly?Phe?Gly?Phe?Val?Asn?Phe?Glu?Asn?Ala?Asp
65 70 75 80
Asp?Ala?Ala?Arg?Ala?Val?Glu?Ser?Leu?Asn?Gly?His?Lys?Phe?Asp?Asp
85 90 95
Lys?Glu?Trp?Tyr?Val?Gly?Arg?Ala?Gln?Lys?Lys?Ser?Glu?Arg?Glu?Thr
100 105 110
Glu?Leu?Arg?Val?Arg?Tyr?Glu?Gln?Asn?Leu?Lys?Glu?Ala?Ala?Asp?Lys
115 120 125
Phe?Gln?Ser?Ser?Asn?Leu?Tyr?Val?Lys?Asn?Leu?Asp?Pro?Ser?Ile?Ser
130 135 140
Asp?Glu?Lys?Leu?Lys?Glu?Ile?Phe?Ser?Pro?Phe?Gly?Thr?Val?Thr?Ser
145 150 155 160
Ser?Lys?Val?Met?Arg?Asp?Pro?Asn?Gly?Thr?Ser?Lys?Gly?Ser?Gly?Phe
165 170 175
Val?Ala?Phe?Ala?Thr?Pro?Glu?Glu?Ala?Thr?Glu?Ala?Met?Ser?Gln?Leu
180 185 190
Ser?Gly?Lys?Met?Ile?Glu?Ser?Lys?Pro?Leu?Tyr?Val?Ala?Ile?Ala?Gln
195 200 205
Arg?Lys?Glu?Asp?Arg?Arg?Val?Arg?Leu?Gln?Ala?Gln?Phe?Ser?Gln?Val
210 215 220
Arg?Pro?Val?Ala?Met?Gln?Pro?Ser?Val?Gly?Pro?Arg?Met?Pro?Val?Tyr
225 230 235 240
Pro?Pro?Gly?Gly?Pro?Gly?Ile?Gly?Gln?Gln?Met?Phe?Tyr?Gly?Gln?Ala
245 250 255
Pro?Pro?Ala?Met?Ile?Pro?Pro?Gln?Pro?Gly?Tyr?Gly?Tyr?Gln?Gln?Gln
260 265 270
Leu?Val?Pro?Gly?Met?Arg?Pro?Gly?Gly?Gly?Pro?Val?Pro?Ser?Phe?Phe
275 280 285
Met?Pro?Met?Val?Gln?Pro?Gln?Gln?Gln?Arg?Pro?Gly?Gly?Gly?Arg?Arg
290 295 300
Pro?Gly?Gly?Ile?Gln?His?Ser?Gln?Gln?Gln?Asn?Pro?Met?Met?Gln?Gln
305 310 315 320
Gln?Met?His?Pro?Arg?Gly?Arg?Met?Phe?Arg?Tyr?Pro?Gln?Gly?Arg?Gly
325 330 335
Gly?Ser?Gly?Asp?Val?Pro?Pro?Tyr?Asp?Met?Gly?Asn?Asn?Met?Pro?Leu
340 345 350
Thr?Ile?Gly?Ala?Leu?Ala?Ser?Asn?Leu?Ser?Asn?Ala?Thr?Pro?Glu?Gln
355 360 365
Gln?Arg?Thr?Met?Leu?Gly?Glu?Val?Leu?Tyr?Pro?Leu?Val?Glu?Gln?Val
370 375 380
Glu?Ala?Glu?Ser?Ala?Ala?Lys?Val?Thr?Gly?Met?Leu?Leu?Glu?Met?Asp
385 390 395 400
Gln?Thr?Glu?Val?Leu?His?Leu?Leu?Glu?Ser?Pro?Glu?Ala?Leu?Lys?Ala
405 410 415
Lys?Val?Ala?Glu?Ala?Met?Asp?Val?Leu?Arg?Ser?Val?Ala?Ala?Gly?Gly
420 425 430
Ala?Thr?Glu?Gln?Leu?Ala?Ser?Leu?Asn?Leu?Ser
435 440
<210>3
<211>1305
<212>DNA
＜213〉Arabidopis thaliana
<400>3
atggcagacg?tcaaggttca?atccgaatcc?gaatcctcgg?attctcatcc?cttggtcgac 60
tatcaatcac?ttccacctta?tcctccgccg?catccaccgg?ttgaagtaga?ggagaatcaa 120
ccaaaaacat?ctccgactcc?gccgccgcca?cactggatgc?gttatccacc?ggtgttaatg 180
cctcagatga?tgtacgcgcc?gccgccaccg?atgccgttct?caccttatca?tcaatatccg 240
aatcaccacc?actttcacca?tcaatctcgt?ggtaataagc?atcaaaacgc?ttttaatggt 300
gagaataaaa?ctatttgggt?tggtgatttg?caaaactgga?tggatgaggc?ttatcttaat 360
tctgctttta?cttccgccga?agagagagag?attgtttcgc?tgaaggtgat?tcgtaataag 420
cacaatggtt?catcggaagg?atatggattt?gtggagtttg?agtcccatga?tgtagctgat 480
aaggttttgc?aggagtttaa?cggggcgcct?atgccaaata?ctgaccaacc?ttttcgtttg 540
aactgggcta?gttttagcac?cggtgagaag?cggttagaga?acaatggacc?tgatctctct 600
atatttgttg?gggatttggc?gccagatgtt?tcggatgctt?tgttgcacga?gaccttctct 660
gagaagtatc?cgtcggttaa?agctgccaaa?gttgtccttg?atgctaatac?tggtagatca 720
aaggggtatg?ggtttgtgag?gtttggagat?gagaatgaaa?ggaccaaagc?aatgactgag 780
atgaatggtg?ttaaatgctc?tagtagagct?atgcgtatcg?gtcctgctac?cccaaggaaa 840
actaatggtt?atcaacaaca?aggtggatac?atgccgagtg?gtgcctttac?gcgttctgaa 900
ggggacacaa?tcaacacaac?aatatttgtt?ggagggcttg?actctagtgt?cactgatgaa 960
gacttaaagc?aacctttctc?tgaattcggg?gaaatagtgt?ctgtcaagat?tcctgttggt 1020
aaaggatgcg?gatttgttca?gtttgttaac?agaccaaatg?cagaggaggc?tttggaaaaa 1080
ctcaatggga?ctgtaattgg?caaacaaaca?gtccggcttt?cttggggccg?taatccagcc 1140
aataagcagc?ctagagataa?gtatggaaac?caatgggttg?atccgtacta?tggaggacag 1200
ttttacaatg?ggtatggata?catggtacct?caacctgacc?cgagaatgta?tcctgctgca 1260
ccttactatc?caatgtacgg?tggtcatcag?caacaagtta?gctga 1305
<210>4
<211>434
<212>PRT
＜213〉Arabidopis thaliana
<400>4
Met?Ala?Asp?Val?Lys?Val?Gln?Ser?Glu?Ser?Glu?Ser?Ser?Asp?Ser?His
1 5 10 15
Pro?Leu?Val?Asp?Tyr?Gln?Ser?Leu?Pro?Pro?Tyr?Pro?Pro?Pro?His?Pro
20 25 30
Pro?Val?Glu?Val?Glu?Glu?Asn?Gln?Pro?Lys?Thr?Ser?Pro?Thr?Pro?Pro
35 40 45
Pro?Pro?His?Trp?Met?Arg?Tyr?Pro?Pro?Val?Leu?Met?Pro?Gln?Met?Met
50 55 60
Tyr?Ala?Pro?Pro?Pro?Pro?Met?Pro?Phe?Ser?Pro?Tyr?His?Gln?Tyr?Pro
65 70 75 80
Asn?His?His?His?Phe?His?His?Gln?Ser?Arg?Gly?Asn?Lys?His?Gln?Asn
85 90 95
Ala?Phe?Asn?Gly?Glu?Asn?Lys?Thr?Ile?Trp?Val?Gly?Asp?Leu?Gln?Asn
100 105 110
Trp?Met?Asp?Glu?Ala?Tyr?Leu?Asn?Ser?Ala?Phe?Thr?Ser?Ala?Glu?Glu
115 120 125
Arg?Glu?Ile?Val?Ser?Leu?Lys?Val?Ile?Arg?Asn?Lys?His?Asn?Gly?Ser
130 135 140
Ser?Glu?Gly?Tyr?Gly?Phe?Val?Glu?Phe?Glu?Ser?His?Asp?Val?Ala?Asp
145 150 155 160
Lys?Val?Leu?Gln?Glu?Phe?Asn?Gly?Ala?Pro?Met?Pro?Asn?Thr?Asp?Gln
165 170 175
Pro?Phe?Arg?Leu?Asn?Trp?Ala?Ser?Phe?Ser?Thr?Gly?Glu?Lys?Arg?Leu
180 185 190
Glu?Asn?Asn?Gly?Pro?Asp?Leu?Ser?Ile?Phe?Val?Gly?Asp?Leu?Ala?Pro
195 200 205
Asp?Val?Ser?Asp?Ala?Leu?Leu?His?Glu?Thr?Phe?Ser?Glu?Lys?Tyr?Pro
210 215 220
Ser?Val?Lys?Ala?Ala?Lys?Val?Val?Leu?Asp?Ala?Asn?Thr?Gly?Arg?Ser
225 230 235 240
Lys?Gly?Tyr?Gly?Phe?Val?Arg?Phe?Gly?Asp?Glu?Asn?Glu?Arg?Thr?Lys
245 250 255
Ala?Met?Thr?Glu?Met?Asn?Gly?Val?Lys?Cys?Ser?Ser?Arg?Ala?Met?Arg
260 265 270
Ile?Gly?Pro?Ala?Thr?Pro?Arg?Lys?Thr?Asn?Gly?Tyr?Gln?Gln?Gln?Gly
275 280 285
Gly?Tyr?Met?Pro?Ser?Gly?Ala?Phe?Thr?Arg?Ser?Glu?Gly?Asp?Thr?Ile
290 295 300
Asn?Thr?Thr?Ile?Phe?Val?Gly?Gly?Leu?Asp?Ser?Ser?Val?Thr?Asp?Glu
305 310 315 320
Asp?Leu?Lys?Gln?Pro?Phe?Ser?Glu?Phe?Gly?Glu?Ile?Val?Ser?Val?Lys
325 330 335
Ile?Pro?Val?Gly?Lys?Gly?Cys?Gly?Phe?Val?Gln?Phe?Val?Asn?Arg?Pro
340 345 350
Asn?Ala?Glu?Glu?Ala?Leu?Glu?Lys?Leu?Asn?Gly?Thr?Val?Ile?Gly?Lys
355 360 365
Gln?Thr?Val?Arg?Leu?Ser?Trp?Gly?Arg?Asn?Pro?Ala?Asn?Lys?Gln?Pro
370 375 380
Arg?Asp?Lys?Tyr?Gly?Asn?Gln?Trp?Val?Asp?Pro?Tyr?Tyr?Gly?Gly?Gln
385 390 395 400
Phe?Tyr?Asn?Gly?Tyr?Gly?Tyr?Met?Val?Pro?Gln?Pro?Asp?Pro?Arg?Met
405 410 415
Tyr?Pro?Ala?Ala?Pro?Tyr?Tyr?Pro?Met?Tyr?Gly?Gly?His?Gln?Gln?Gln
420 425 430
Val?Ser
<210>5
<211>369
<212>DNA
＜213〉Arabidopis thaliana
<400>5
atggcgtatg?aaccgatgaa?gcccacgaaa?gctggtttgg?aggctcctct?ggagcagatt 60
cataagatca?ggatcactct?ctcttcaaaa?aatgtgaaga?acttggaaaa?agtgtgcact 120
gatttggtcc?gtggagctaa?ggataagaga?cttagagtta?agggaccagt?gagaatgccc 180
actaaggttc?ttaagatcac?taccagaaag?gcaccttgtg?gtgaaggtac?caatacttgg 240
gacaggtttg?agctcagggt?tcacaagcgt?gtcatcgatc?tcttcagctc?ccctgacgtt 300
gttaagcaaa?tcacgtctat?caccattgag?cccggtgttg?aggtcgaggt?cactattgct 360
gactcttag 369
<210>6
<211>122
<212>PRT
＜213〉Arabidopis thaliana
<400>6
Met?Ala?Tyr?Glu?Pro?Met?Lys?Pro?Thr?Lys?Ala?Gly?Leu?Glu?Ala?Pro
1 5 10 15
Leu?Glu?Gln?Ile?His?Lys?Ile?Arg?Ile?Thr?Leu?Ser?Ser?Lys?Asn?Val
20 25 30
Lys?Asn?Leu?Glu?Lys?Val?Cys?Thr?Asp?Leu?Val?Arg?Gly?Ala?Lys?Asp
35 40 45
Lys?Arg?Leu?Arg?Val?Lys?Gly?Pro?Val?Arg?Met?Pro?Thr?Lys?Val?Leu
50 55 60
Lys?Ile?Thr?Thr?Arg?Lys?Ala?Pro?Cys?Gly?Glu?Gly?Thr?Asn?Thr?Trp
65 70 75 80
Asp?Arg?Phe?Glu?Leu?Arg?Val?His?Lys?Arg?Val?Ile?Asp?Leu?Phe?Ser
85 90 95
Ser?Pro?Asp?Val?Val?Lys?Gln?Ile?Thr?Ser?Ile?Thr?Ile?Glu?Pro?Gly
100 105 110
Val?Glu?Val?Glu?Val?Thr?Ile?Ala?Asp?Ser
115 120
<210>7
<211>741
<212>DNA
＜213〉Arabidopis thaliana
<400>7
atggggagaa?gaccatgctg?tgagaagata?ggattgaaga?aagggccatg?gagtgctgaa 60
gaagatcgaa?tcttgatcaa?ttatattagt?ctccatggcc?atcccaattg?gagagctctc 120
cctaaactag?ccgggctact?tcggtgcgga?aaaagttgca?ggcttcgttg?gattaattat 180
ttgagaccag?acatcaaacg?tggcaatttc?actcctcatg?aagaagatac?tatcatcagc 240
ttacatcaac?tcttaggcaa?cagatggtct?gcgatagctg?caaaattgcc?tggacgaaca 300
gacaacgaaa?ttaaaaatgt?ttggcacact?catttaaaga?aaagactcca?ccacagtcaa 360
gatcaaaaca?acaaggaaga?tttcgtctct?actacagctg?cggagatgcc?aacctctccg 420
caacaacaat?ctagtagtag?tgccgacatt?tcagcaatta?caacattggg?aaacaacaat 480
gacatctcca?atagcaacaa?agactccgcg?acgtcatccg?aagatgttct?tgcaattata 540
gatgagagct?tttggtcaga?agtggtattg?atggactgtg?acatttcagg?aaatgagaag 600
aatgagaaaa?agatagagaa?ttgggagggc?tcactagata?gaaacgataa?gggatataac 660
catgacatgg?agttttggtt?tgaccatctc?actagtagta?gttgtataat?tggagaaatg 720
tccgacattt?ctgagttttg?a 741
<210>8
<211>246
<212>PRT
＜213〉Arabidopis thaliana
<400>8
Met?Gly?Arg?Arg?Pro?Cys?Cys?Glu?Lys?Ile?Gly?Leu?Lys?Lys?Gly?Pro
1 5 10 15
Trp?Ser?Ala?Glu?Glu?Asp?Arg?Ile?Leu?Ile?Asn?Tyr?Ile?Ser?Leu?His
20 25 30
Gly?His?Pro?Asn?Trp?Arg?Ala?Leu?Pro?Lys?Leu?Ala?Gly?Leu?Leu?Arg
35 40 45
Cys?Gly?Lys?Ser?Cys?Arg?Leu?Arg?Trp?Ile?Asn?Tyr?Leu?Arg?Pro?Asp
50 55 60
Ile?Lys?Arg?Gly?Asn?Phe?Thr?Pro?His?Glu?Glu?Asp?Thr?Ile?Ile?Ser
65 70 75 80
Leu?His?Gln?Leu?Leu?Gly?Asn?Arg?Trp?Ser?Ala?lle?Ala?Ala?Lys?Leu
85 90 95
Pro?Gly?Arg?Thr?Asp?Asn?Glu?Ile?Lys?Asn?Val?Trp?His?Thr?His?Leu
100 105 110
Lys?Lys?Arg?Leu?His?His?Ser?Gln?Asp?Gln?Asn?Asn?Lys?Glu?Asp?Phe
115 120 125
Val?Ser?Thr?Thr?Ala?Ala?Glu?Met?Pro?Thr?Ser?Pro?Gln?Gln?Gln?Ser
130 135 140
Ser?Ser?Ser?Ala?Asp?Ile?Ser?Ala?Ile?Thr?Thr?Leu?Gly?Asn?Asn?Asn
145 150 155 160
Asp?Ile?Ser?Asn?Ser?Asn?Lys?Asp?Ser?Ala?Thr?Ser?Ser?Glu?Asp?Val
165 170 175
Leu?Ala?Ile?Ile?Asp?Glu?Ser?Phe?Trp?Ser?Glu?Val?Val?Leu?Met?Asp
180 185 190
Cys?Asp?Ile?Ser?Gly?Asn?Glu?Lys?Asn?Glu?Lys?Lys?Ile?Glu?Asn?Trp
195 200 205
Glu?Gly?Ser?Leu?Asp?Arg?Asn?Asp?Lys?Gly?Tyr?Asn?His?Asp?Met?Glu
210 215 220
Phe?Trp?Phe?Asp?His?Leu?Thr?Ser?Ser?Ser?Cys?Ile?Ile?Gly?Glu?Met
225 230 235 240
Ser?Asp?Ile?Ser?Glu?Phe
245
<210>9
<211>1125
<212>DNA
＜213〉Arabidopis thaliana
<400>9
atgggaagag?caccgtgttg?tgataaggcc?aacgtgaaga?aagggccttg?gtctcctgag 60
gaagacgcca?aactcaaaga?ttacatcgag?aatagtggca?caggaggcaa?ctggattgct 120
ttgcctcaga?aaattggttt?aaggagatgt?gggaagagtt?gcaggctaag?gtggctcaac 180
tatttgagac?caaacatcaa?acatggtggc?ttctccgagg?aagaagacaa?catcatttgt 240
aacctctatg?ttactattgg?tagcaggtgg?tctataattg?ctgcacaatt?gccgggaaga 300
accgacaacg?atatcaaaaa?ctattggaac?acgaggctga?agaagaagct?tctgaacaaa 360
caaaggaaag?agttccaaga?agcgcgaatg?aagcaagaga?tggtgatgat?gaaaaggcaa 420
caacaaggac?aaggacaagg?tcaaagtaat?ggtagtacgg?atctttatct?taacaacatg 480
tttggatcat?caccatggcc?attactacca?caacttcctc?ctccacatca?tcaaatacct 540
cttggaatga?tggaaccaac?aagctgtaac?tactaccaaa?cgacaccgtc?ttgtaaccta 600
gaacaaaagc?cattgatcac?actcaagaac?atggtcaaga?ttgaagaaga?acaggaaagg 660
acaaaccctg?atcatcatca?tcaagattct?gtcacaaacc?cttttgattt?ctctttctct 720
cagcttttgt?tagatcccaa?ttactatctg?ggatcaggag?ggggaggaga?aggagatttt 780
gctatcatga?gcagcagcac?aaactcacca?ttaccaaaca?caagtagtga?tcaacatcca 840
agtcaacagc?aagagattct?tcaatggttt?gggagcagta?actttcagac?agaagcaatc 900
aacgatatgt?tcataaacaa?caacaacaac?atagtgaatc?ttgagaccat?cgagaacaca 960
aaagtctatg?gagacgcctc?agtagccgga?gccgctgtcc?gagcagcttt?gggcggaggg 1020
acaacgagta?catcggcgga?tcaaagtaca?ataagttggg?aggatataac?ttctctagtt 1080
aattccgaag?atgcaagtta?cttcaatgcg?ccaaatcatg?tgtaa 1125
<210>10
<211>374
<212>PRT
＜213〉Arabidopis thaliana
<400>10
Met?Gly?Arg?Ala?Pro?Cys?Cys?Asp?Lys?Ala?Asn?Val?Lys?Lys?Gly?Pro
1 5 10 15
Trp?Ser?Pro?Glu?Glu?Asp?Ala?Lys?Leu?Lys?Asp?Tyr?Ile?Glu?Asn?Ser
20 25 30
Gly?Thr?Gly?Gly?Asn?Trp?Ile?Ala?Leu?Pro?Gln?Lys?Ile?Gly?Leu?Arg
35 40 45
Arg?Cys?Gly?Lys?Ser?Cys?Arg?Leu?Arg?Trp?Leu?Asn?Tyr?Leu?Arg?Pro
50 55 60
Asn?Ile?Lys?His?Gly?Gly?Phe?Ser?Glu?Glu?Glu?Asp?Asn?Ile?Ile?Cys
65 70 75 80
Asn?Leu?Tyr?Val?Thr?Ile?Gly?Ser?Arg?Trp?Ser?Ile?Ile?Ala?Ala?Gln
85 90 95
Leu?Pro?Gly?Arg?Thr?Asp?Asn?Asp?Ile?Lys?Asn?Tyr?Trp?Asn?Thr?Arg
100 105 110
Leu?Lys?Lys?Lys?Leu?Leu?Asn?Lys?Gln?Arg?Lys?Glu?Phe?Gln?Glu?Ala
115 120 125
Arg?Met?Lys?Gln?Glu?Met?Val?Met?Met?Lys?Arg?Gln?Gln?Gln?Gly?Gln
130 135 140
Gly?Gln?Gly?Gln?Ser?Asn?Gly?Ser?Thr?Asp?Leu?Tyr?Leu?Asn?Asn?Met
145 150 155 160
Phe?Gly?Ser?Ser?Pro?Trp?Pro?Leu?Leu?Pro?Gln?Leu?Pro?Pro?Pro?His
165 170 175
His?Gln?Ile?Pro?Leu?Gly?Met?Met?Glu?Pro?Thr?Ser?Cys?Asn?Tyr?Tyr
180 185 190
Gln?Thr?Thr?Pro?Ser?Cys?Asn?Leu?Glu?Gln?Lys?Pro?Leu?Ile?Thr?Leu
195 200 205
Lys?Asn?Met?Val?Lys?Ile?Glu?Glu?Glu?Gln?Glu?Arg?Thr?Asn?Pro?Asp
210 215 220
His?His?His?Gln?Asp?Ser?Val?Thr?Asn?Pro?Phe?Asp?Phe?Ser?Phe?Ser
225 230 235 240
Gln?Leu?Leu?Leu?Asp?Pro?Asn?Tyr?Tyr?Leu?Gly?Ser?Gly?Gly?Gly?Gly
245 250 255
Glu?Gly?Asp?Phe?Ala?Ile?Met?Ser?Ser?Ser?Thr?Asn?Ser?Pro?Leu?Pro
260 265 270
Asn?Thr?Ser?Ser?Asp?Gln?His?Pro?Ser?Gln?Gln?Gln?Glu?Ile?Leu?Gln
275 280 285
Trp?Phe?Gly?Ser?Ser?Asn?Phe?Gln?Thr?Glu?Ala?Ile?Asn?Asp?Met?Phe
290 295 300
Ile?Asn?Asn?Asn?Asn?Asn?Ile?Val?Asn?Leu?Glu?Thr?Ile?Glu?Asn?Thr
305 310 315 320
Lys?Val?Tyr?Gly?Asp?Ala?Ser?Val?Ala?Gly?Ala?Ala?Val?Arg?Ala?Ala
325 330 335
Leu?Gly?Gly?Gly?Thr?Thr?Ser?Thr?Ser?Ala?Asp?Gln?Ser?Thr?Ile?Ser
340 345 350
Trp?Glu?Asp?Ile?Thr?Ser?Leu?Val?Asn?Ser?Glu?Asp?Ala?Ser?Tyr?Phe
355 360 365
Asn?Ala?Pro?Asn?His?Val
370
<210>11
<211>1164
<212>DNA
＜213〉Arabidopis thaliana
<400>11
atgcagcaac?caccgtcaaa?cgccgccgga?gctggacaga?taccatcagg?acaacagcat 60
ttgtggatga?tgatgcaaca?gcagcagcag?cagcagcaga?tgcagttgtc?tgcggcgcca 120
ctaggtcaac?atcagtacgg?tattggatct?cagaatccag?gatccgctag?cgatgttaag 180
tcgttgtgga?tcggagactt?gcagcaatgg?atggacgaga?actacatcat?gagcgtcttt 240
gctcagtctg?gcgaggctac?atcagctaaa?gtcattcgta?ataagctgac?gggacaatct 300
gaaggttatg?gattcattga?gttcgtcagc?cactctgtag?cagagcgggt?tttgcagact 360
tacaatggtg?ctcccatgcc?gagcactgaa?cagacgttta?ggctcaactg?ggctcaggct 420
ggggctggag?agaaacgatt?ccagactgaa?gggcctgacc?ataccatttt?cgtaggtgac 480
ttggcacctg?aggtgactga?ctatatgctc?tcggacacat?tcaagaatgt?gtatgggtct 540
gtcaaagggg?ctaaagttgt?gcttgacagg?accactggaa?ggtccaaggg?gtatgggttt 600
gttaggtttg?cggatgaaaa?tgagcagatg?cgtgccatga?ctgaaatgaa?tggtcaatac 660
tgctcgacaa?ggcctatgcg?tattggtccg?gctgccaata?agaatgctct?tccgatgcaa 720
ccagctatgt?atcaaaacac?tcaaggagca?aatgctggag?ataatgatcc?taataacaca 780
acaatttttg?ttggaggtct?ggatgctaat?gttacagacg?atgaattaaa?gtcaattttt 840
ggtcaatttg?gtgaacttct?tcatgtgaaa?atacctccag?gaaaacgttg?tggattcgtt 900
caatatgcca?acaaggcgtc?tgcagagcat?gcactttcgg?tgctgaatgg?aacacaatta 960
ggtggacaaa?gcatccgtct?ttcgtgggga?cgtagtccaa?acaagcagtc?tgatcaagcg 1020
caatggaacg?gtggtggata?ctatggatac?cctccacagc?cacagggcgg?ctatggttat 1080
gcagctcaac?caccaactca?agaccctaat?gcgtactatg?gtggttacac?tggctatggc 1140
aactatcagc?agcaacgtca?gtga 1164
<210>12
<211>387
<212>PRT
＜213〉Arabidopis thaliana
<400>12
Met?Gln?Gln?Pro?Pro?Ser?Asn?Ala?Ala?Gly?Ala?Gly?Gln?Ile?Pro?Ser
1 5 10 15
Gly?Gln?Gln?His?Leu?Trp?Met?Met?Met?Gln?Gln?Gln?Gln?Gln?Gln?Gln
20 25 30
Gln?Met?Gln?Leu?Ser?Ala?Ala?Pro?Leu?Gly?Gln?His?Gln?Tyr?Gly?Ile
35 40 45
Gly?Ser?Gln?Asn?Pro?Gly?Ser?Ala?Ser?Asp?Val?Lys?Ser?Leu?Trp?Ile
50 55 60
Gly?Asp?Leu?Gln?Gln?Trp?Met?Asp?Glu?Asn?Tyr?Ile?Met?Ser?Val?Phe
65 70 75 80
Ala?Gln?Ser?Gly?Glu?Ala?Thr?Ser?Ala?Lys?Val?Ile?Arg?Asn?Lys?Leu
85 90 95
Thr?Gly?Gln?Ser?Glu?Gly?Tyr?Gly?Phe?Ile?Glu?Phe?Val?Ser?His?Ser
100 105 110
Val?Ala?Glu?Arg?Val?Leu?Gln?Thr?Tyr?Asn?Gly?Ala?Pro?Met?Pro?Ser
115 120 125
Thr?Glu?Gln?Thr?Phe?Arg?Leu?Asn?Trp?Ala?Gln?Ala?Gly?Ala?Gly?Glu
130 135 140
Lys?Arg?Phe?Gln?Thr?Glu?Gly?Pro?Asp?His?Thr?Ile?Phe?Val?Gly?Asp
145 150 155 160
Leu?Ala?Pro?Glu?Val?Thr?Asp?Tyr?Met?Leu?Ser?Asp?Thr?Phe?Lys?Asn
165 170 175
Val?Tyr?Gly?Ser?Val?Lys?Gly?Ala?Lys?Val?Val?Leu?Asp?Arg?Thr?Thr
180 185 190
Gly?Arg?Ser?Lys?Gly?Tyr?Gly?Phe?Val?Arg?Phe?Ala?Asp?Glu?Asn?Glu
195 200 205
Gln?Met?Arg?Ala?Met?Thr?Glu?Met?Asn?Gly?Gln?Tyr?Cys?Ser?Thr?Arg
210 215 220
Pro?Met?Arg?Ile?Gly?Pro?Ala?Ala?Asn?Lys?Asn?Ala?Leu?Pro?Met?Gln
225 230 235 240
Pro?Ala?Met?Tyr?Gln?Asn?Thr?Gln?Gly?Ala?Asn?Ala?Gly?Asp?Asn?Asp
245 250 255
Pro?Asn?Asn?Thr?Thr?Ile?Phe?Val?Gly?Gly?Leu?Asp?Ala?Asn?Val?Thr
260 265 270
Asp?Asp?Glu?Leu?Lys?Ser?Ile?Phe?Gly?Gln?Phe?Gly?Glu?Leu?Leu?His
275 280 285
Val?Lys?Ile?Pro?Pro?Gly?Lys?Arg?Cys?Gly?Phe?Val?Gln?Tyr?Ala?Asn
290 295 300
Lys?Ala?Ser?Ala?Glu?His?Ala?Leu?Ser?Val?Leu?Asn?Gly?Thr?Gln?Leu
305 310 315 320
Gly?Gly?Gln?Ser?Ile?Arg?Leu?Ser?Trp?Gly?Arg?Ser?Pro?Asn?Lys?Gln
325 330 335
Ser?Asp?Gln?Ala?Gln?Trp?Asn?Gly?Gly?Gly?Tyr?Tyr?Gly?Tyr?Pro?Pro
340 345 350
Gln?Pro?Gln?Gly?Gly?Tyr?Gly?Tyr?Ala?Ala?Gln?Pro?Pro?Thr?Gln?Asp
355 360 365
Pro?Asn?Ala?Tyr?Tyr?Gly?Gly?Tyr?Thr?Gly?Tyr?Gly?Asn?Tyr?Gln?Gln
370 375 380
Gln?Arg?Gln
385
<210>13
<211>1218
<212>DNA
＜213〉Arabidopis thaliana
<400>13
atgatgcagc?agccaccacc?cggaggtatc?cttccacatc?acgctcctcc?tccttctgcg 60
caacaacagt?acggttacca?acaaccttac?gggattgctg?gagctgctcc?accaccacca 120
cagatgtgga?atcctcaagc?ggcggcgccg?ccatcagttc?agcctacgac?cgctgacgag 180
atccggactc?tttggatcgg?ggacttacag?tattggatgg?atgagaattt?cctctacggt 240
tgctttgctc?ataccggaga?gatggtttct?gctaaagtga?ttcgtaacaa?gcaaaccggt 300
caagttgaag?gatacggttt?cattgaattc?gcatctcatg?ctgctgctga?aagagttcta 360
caaacattca?acaacgctcc?tatcccgagc?tttcctgatc?agctctttag?actgaactgg 420
gcatcattga?gttcaggaga?taaacgagac?gattcaccgg?actacacgat?atttgtcggt 480
gatctggctg?ctgatgttac?ggattatatc?ttacttgaga?cgttcagagcc?tcttatccg 540
tcagtgaagg?gtgcaaaggt?tgttattgac?agagtcactg?gacgtacaaa?aggatatggg 600
tttgttaggt?tttctgatga?aagtgaacag?atccgtgcta?tgacggagat?gaatggcgtt 660
ccttgttcta?ctagacctat?gagaattggt?cccgctgcta?gcaagaaagg?tgtaactggt 720
caaagagatt?cataccagag?ctctgctgca?ggggtaacaa?ctgataatga?tccaaataac 780
acaactgttt?ttgttggtgg?attagatgca?tctgtcacgg?atgatcatct?gaagaatgtc 840
tttagccaat?atggtgagat?tgtgcatgtg?aaaatacccg?ctggaaagcg?ctgtggattc 900
gttcagtttt?ccgagaagag?ctgtgcagag?gaagctctta?gaatgctgaa?tggagtgcaa 960
ttaggcggaa?caaccgtcag?gctctcatgg?ggccgaagtc?cttcgaacaa?acagtcgggg 1020
gatccgagcc?agttttacta?cggtgggtat?ggacaaggac?aggagcagta?tgggtacacg 1080
atgcctcaag?accctaatgc?atattacgga?ggctactctg?gtggaggata?cagcggtggt 1140
taccagcaga?caccacaggc?aggacagcaa?ccaccacaac?agccaccaca?gcagcaacaa 1200
gtcgggttta?gctactaa 1218
<210>14
<211>405
<212>PRT
＜213〉Arabidopis thaliana
<400>14
Met?Met?Gln?Gln?Pro?Pro?Pro?Gly?Gly?Ile?Leu?Pro?His?His?Ala?Pro
1 5 10 15
Pro?Pro?Ser?Ala?Gln?Gln?Gln?Tyr?Gly?Tyr?Gln?Gln?Pro?Tyr?Gly?Ile
20 25 30
Ala?Gly?Ala?Ala?Pro?Pro?Pro?Pro?Gln?Met?Trp?Asn?Pro?Gln?Ala?Ala
35 40 45
Ala?Pro?Pro?Ser?Val?Gln?Pro?Thr?Thr?Ala?Asp?Glu?Ile?Arg?Thr?Leu
50 55 60
Trp?Ile?Gly?Asp?Leu?Gln?Tyr?Trp?Met?Asp?Glu?Asn?Phe?Leu?Tyr?Gly
65 70 75 80
Cys?Phe?Ala?His?Thr?Gly?Glu?Met?Val?Ser?Ala?Lys?Val?Ile?Arg?Asn
85 90 95
Lys?Gln?Thr?Gly?Gln?Val?Glu?Gly?Tyr?Gly?Phe?Ile?Glu?Phe?Ala?Ser
100 105 110
His?Ala?Ala?Ala?Glu?Arg?Val?Leu?Gln?Thr?Phe?Asn?Asn?Ala?Pro?Ile
115 120 125
Pro?Ser?Phe?Pro?Asp?Gln?Leu?Phe?Arg?Leu?Asn?Trp?Ala?Ser?Leu?Ser
130 135 140
Ser?Gly?Asp?Lys?Arg?Asp?Asp?Ser?Pro?Asp?Tyr?Thr?Ile?Phe?Val?Gly
145 150 155 160
Asp?Leu?Ala?Ala?Asp?Val?Thr?Asp?Tyr?Ile?Leu?Leu?Glu?Thr?Phe?Arg
165 170 175
Ala?Ser?Tyr?Pro?Ser?Val?Lys?Gly?Ala?Lys?Val?Val?Ile?Asp?Arg?Val
180 185 190
Thr?Gly?Arg?Thr?Lys?Gly?Tyr?Gly?Phe?Val?Arg?Phe?Ser?Asp?Glu?Ser
195 200 205
Glu?Gln?Ile?Arg?Ala?Met?Thr?Glu?Met?Asn?Gly?Val?Pro?Cys?Ser?Thr
210 215 220
Arg?Pro?Met?Arg?Ile?Gly?Pro?Ala?Ala?Ser?Lys?Lys?Gly?Val?Thr?Gly
225 230 235 240
Gln?Arg?Asp?Ser?Tyr?Gln?Ser?Ser?Ala?Ala?Gly?Val?Thr?Thr?Asp?Asn
245 250 255
Asp?Pro?Asn?Asn?Thr?Thr?Val?Phe?Val?Gly?Gly?Leu?Asp?Ala?Ser?Val
260 265 270
Thr?Asp?Asp?His?Leu?Lys?Asn?Val?Phe?Ser?Gln?Tyr?Gly?Glu?Ile?Val
275 280 285
His?Val?Lys?Ile?Pro?Ala?Gly?Lys?Arg?Cys?Gly?Phe?Val?Gln?Phe?Ser
290 295 300
Glu?Lys?Ser?Cys?Ala?Glu?Glu?Ala?Leu?Arg?Met?Leu?Asn?Gly?Val?Gln
305 310 315 320
Leu?Gly?Gly?Thr?Thr?Val?Arg?Leu?Ser?Trp?Gly?Arg?Ser?Pro?Ser?Asn
325 330 335
Lys?Gln?Ser?Gly?Asp?Pro?Ser?Gln?Phe?Tyr?Tyr?Gly?Gly?Tyr?Gly?Gln
340 345 350
Gly?Gln?Glu?Gln?Tyr?Gly?Tyr?Thr?Met?Pro?Gln?Asp?Pro?Asn?Ala?Tyr
355 360 365
Tyr?Gly?Gly?Tyr?Ser?Gly?Gly?Gly?Tyr?Ser?Gly?Gly?Tyr?Gln?Gln?Thr
370 375 380
Pro?Gln?Ala?Gly?Gln?Gln?Pro?Pro?Gln?Gln?Pro?Pro?Gln?Gln?Gln?Gln
385 390 395 400
Val?Gly?Phe?Ser?Tyr
405
<210>15
<211>1248
<212>DNA
＜213〉Arabidopis thaliana
<400>15
atgatgcagc?agccacctcc?agcttccaac?ggtgctgcaa?cagggccagg?gcagattcct 60
tccgaccaac?aagcttacct?ccagcagcag?cagtcgtgga?tgatgcagca?ccagcagcaa 120
caacaaggtc?agccgcctgc?aggatggaat?cagcagtctg?caccgtcttc?tggtcaacca 180
cagcagcagc?agtatggtgg?tggtggatct?cagaatccag?gatcagctgg?tgagatccgg 240
tccctgtgga?tcggtgactt?gcagccatgg?atggatgaga?actatctcat?gaacgtcttt 300
ggtcttactg?gcgaggctac?agcagctaaa?gttattcgca?ataaacagaa?cggatattca 360
gaaggttatg?gctttattga?gtttgtgaac?catgctacag?ctgagaggaa?tttacagact 420
tacaatggtg?ctccgatgcc?gagcagtgag?caggccttca?ggttgaactg?ggctcagctt 480
ggagctggag?agagacgcca?ggctgaaggg?cctgagcaca?cagtttttgt?tggagacttg 540
gcacctgatg?ttaccgacca?catgcttact?gaaacgttta?aagctgtgta?ttcctctgtc 600
aagggagcta?aagttgtgaa?tgataggact?actggacggt?ccaagggtta?tggatttgtc 660
aggtttgcgg?atgaaagtga?gcagattcgt?gceatgactg?aaatgaatgg?tcaatactgc 720
tcatcaaggc?ctatgcgtac?tggtcctgct?gccaacaaga?agcctcttac?aatgcaacca 780
gcttcatatc?agaacactca?aggaaattca?ggagaaagtg?atccaactaa?cacaacaatt 840
tttgttggag?ctgtggatca?aagtgtaaca?gaagatgatt?tgaagtcagt?ttttggtcaa 900
tttggtgaac?tagttcatgt?gaaaataccc?gcaggaaaac?gttgcggatt?tgttcaatac 960
gccaataggg?catgtgctga?gcaagcactt?tctgtgttga?acggaacaca?acttggggga 1020
caaagcattc?gtctttcatg?gggtcgcagt?ccttccaaca?aacagactca?acctgatcaa 1080
gcccagtatg?gtggtggtgg?aggatactat?gggtatcctc?ctcaaggata?tgaagcatac 1140
ggatatgcac?ctcctcctca?ggaccctaac?gcctactacg?gtggttatgc?tgggggcggc 1200
tatggaaact?accagcagcc?tggtggatac?cagcagcaac?agcagtga 1248
<210>16
<211>415
<212>PRT
＜213〉Arabidopis thaliana
<400>16
Met?Met?Gln?Gln?Pro?Pro?Pro?Ala?Ser?Asn?Gly?Ala?Ala?Thr?Gly?Pro
1 5 10 15
Gly?Gln?Ile?Pro?Ser?Asp?Gln?Gln?Ala?Tyr?Leu?Gln?Gln?Gln?Gln?Ser
20 25 30
Trp?Met?Met?Gln?His?Gln?Gln?Gln?Gln?Gln?Gly?Gln?Pro?Pro?Ala?Gly
35 40 45
Trp?Asn?Gln?Gln?Ser?Ala?Pro?Ser?Ser?Gly?Gln?Pro?Gln?Gln?Gln?Gln
50 55 60
Tyr?Gly?Gly?Gly?Gly?Ser?Gln?Asn?Pro?Gly?Ser?Ala?Gly?Glu?Ile?Arg
65 70 75 80
Ser?Leu?Trp?Ile?Gly?Asp?Leu?Gln?Pro?Trp?Met?Asp?Glu?Asn?Tyr?Leu
85 90 95
Met?Asn?Val?Phe?Gly?Leu?Thr?Gly?Glu?Ala?Thr?Ala?Ala?Lys?Val?Ile
100 105 110
Arg?Asn?Lys?Gln?Asn?Gly?Tyr?Ser?Glu?Gly?Tyr?Gly?Phe?lle?Glu?Phe
115 120 125
Val?Asn?His?Ala?Thr?Ala?Glu?Arg?Asn?Leu?Gln?Thr?Tyr?Asn?Gly?Ala
130 135 240
Pro?Met?Pro?Ser?Ser?Glu?Gln?Ala?Phe?Arg?Leu?Asn?Trp?Ala?Gln?Leu
145 150 155 160
Gly?Ala?Gly?Glu?Arg?Arg?Gln?Ala?Glu?Gly?Pro?Glu?His?Thr?Val?Phe
165 170 175
Val?Gly?Asp?Leu?Ala?Pro?Asp?Val?Thr?Asp?His?Met?Leu?Thr?Glu?Thr
180 185 190
Phe?Lys?Ala?Val?Tyr?Ser?Ser?Val?Lys?Gly?Ala?Lys?Val?Val?Asn?Asp
195 200 205
Arg?Thr?Thr?Gly?Arg?Ser?Lys?Gly?Tyr?Gly?Phe?Val?Arg?Phe?Ala?Asp
210 215 220
Glu?Ser?Glu?Gln?Ile?Arg?Ala?Met?Thr?Glu?Met?Asn?Gly?Gln?Tyr?Cys
225 230 235 240
Ser?Ser?Arg?Pro?Met?Arg?Thr?Gly?Pro?Ala?Ala?Asn?Lys?Lys?Pro?Leu
245 250 255
Thr?Met?Gln?Pro?Ala?Ser?Tyr?Gln?Asn?Thr?Gln?Gly?Asn?Ser?Gly?Glu
260 265 270
Ser?Asp?Pro?Thr?Asn?Thr?Thr?Ile?Phe?Val?Gly?Ala?Val?Asp?Gln?Ser
275 280 285
Val?Thr?Glu?Asp?Asp?Leu?Lys?Ser?Val?Phe?Gly?Gln?Phe?Gly?Glu?Leu
290 295 300
Val?His?Val?Lys?Ile?Pro?Ala?Gly?Lys?Arg?Cys?Gly?Phe?Val?Gln?Tyr
305 310 315 320
Ala?Asn?Arg?Ala?Cys?Ala?Glu?Gln?Ala?Leu?Ser?Val?Leu?Asn?Gly?Thr
325 330 335
Gln?Leu?Gly?Gly?Gln?Ser?Ile?Arg?Leu?Ser?Trp?Gly?Arg?Ser?Pro?Ser
340 345 350
Asn?Lys?Gln?Thr?Gln?Pro?Asp?Gln?Ala?Gln?Tyr?Gly?Gly?Gly?Gly?Gly
355 360 365
Tyr?Tyr?Gly?Tyr?Pro?Pro?Gln?Gly?Tyr?Glu?Ala?Tyr?Gly?Tyr?Ala?Pro
370 375 380
Pro?Pro?Gln?Asp?Pro?Asn?Ala?Tyr?Tyr?Gly?Gly?Tyr?Ala?Gly?Gly?Gly
385 390 395 400
Tyr?Gly?Asn?Tyr?Gln?Gln?Pro?Gly?Gly?Tyr?Gln?Gln?Gln?Gln?Gln
405 410 415
<210>17
<211>1278
<212>DNA
＜213〉Arabidopis thaliana
<400>17
atggcgatga?tgcatcctcc?gcagccgccg?caaggctcct?atcaccatcc?tcagacgctc 60
gaagaagttc?gaactctttg?gattggtgat?ttgcagtact?gggtcgacga?aaattacctc 120
acttcctgct?tctcccaaac?cggcgagctc?gtttctgtca?aggtaatacg?taacaagatc 180
acgggacagc?cagaggggta?tggttttata?gagtttatat?ctcatgcagc?agcagagaga 240
actctgcaga?cgtacaatgg?gacacagatg?cctggaactg?agttaacttt?tcggttaaat 300
tgggcttctt?ttggttcagg?acagaaagtt?gatgctggac?ctgatcattc?tatctttgtt 360
ggagatttag?cacctgatgt?tacagattat?cttcttcaag?agacattccg?tgttcattat 420
tcttctgtta?gaggtgccaa?ggttgttact?gatccaagta?ctggacgatc?aaagggttat 480
ggatttgtaa?aatttgcaga?ggaaagtgaa?aggaatcggg?ctatggctga?aatgaatggt 540
ttgtattgct?caacaaggcc?tatgcgtatt?agcgcagcaa?cacctaaaaa?aaacgtcggt 600
gtgcagcaac?aatatgtcac?caaagctgtt?tacccagtta?cagtcccatc?tgcagttgct 660
gcaccagtcc?aagcatacgt?tgctccacct?gaaagtgatg?tcacctgtac?aacgatttca 720
gttgccaatt?tggaccaaaa?tgttacagag?gaagagctga?agaaagcatt?ctcccaatta 780
ggagaggtta?tttatgtcaa?aatacctgca?acaaagggat?atggttatgt?tcaattcaaa 840
accaggcctt?ctgcagaaga?agctgttcaa?agaatgcagg?gacaagtgat?tggtcaacaa 900
gcagttcgca?tctcttggag?taaaaatcca?ggacaggatg?gttgggttac?acaagcagat 960
ccgaatcagt?ggaatgggta?ttatggttat?gggcaaggct?atgatgcata?tgcttatggg 1020
gcaactcaag?atccatccgt?gtacgcatat?ggtggatatg?gctatcccca?gtatccgcaa 1080
cagggagagg?gtacacaaga?catttcgaac?tctgcggcgg?gtggagtagc?aggtgcagag 1140
caagagttgt?atgatcctct?ggccactcct?gatgtagaca?agttaaatgc?tgcttacctt 1200
tcggttcatg?caagtgccat?attaggaagg?ccaatgtggc?agcggacctc?atcgctcaca 1260
tcacaattgg?gcaaatga 1278
<210>18
<211>425
<212>PRT
＜213〉Arabidopis thaliana
<400>18
Met?Ala?Met?Met?His?Pro?Pro?Gln?Pro?Pro?Gln?Gly?Ser?Tyr?His?His
1 5 10 15
Pro?Gln?Thr?Leu?Glu?Glu?Val?Arg?Thr?Leu?Trp?Ile?Gly?Asp?Leu?Gln
20 25 30
Tyr?Trp?Val?Asp?Glu?Asn?Tyr?Leu?Thr?Ser?Cys?Phe?Ser?Gln?Thr?Gly
35 40 45
Glu?Leu?Val?Ser?Val?Lys?Val?Ile?Arg?Asn?Lys?Ile?Thr?Gly?Gln?Pro
50 55 60
Glu?Gly?Tyr?Gly?Phe?Ile?Glu?Phe?Ile?Ser?His?Ala?Ala?Ala?Glu?Arg
65 70 75 80
Thr?Leu?Gln?Thr?Tyr?Asn?Gly?Thr?Gln?Met?Pro?Gly?Thr?Glu?Leu?Thr
85 90 95
Phe?Arg?Leu?Asn?Trp?Ala?Ser?Phe?Gly?Ser?Gly?Gln?Lys?Val?Asp?Ala
100 105 110
Gly?Pro?Asp?His?Ser?Ile?Phe?Val?Gly?Asp?Leu?Ala?Pro?Asp?Val?Thr
115 120 125
Asp?Tyr?Leu?Leu?Gln?Glu?Thr?Phe?Arg?Val?His?Tyr?Ser?Ser?Val?Arg
130 135 140
Gly?Ala?Lys?Val?Val?Thr?Asp?Pro?Ser?Thr?Gly?Arg?Ser?Lys?Gly?Tyr
145 150 155 160
Gly?Phe?Val?Lys?Phe?Ala?Glu?Glu?Ser?Glu?Arg?Asn?Arg?Ala?Met?Ala
165 170 175
Glu?Met?Asn?Gly?Leu?Tyr?Cys?Ser?Thr?Arg?Pro?Met?Arg?Ile?Ser?Ala
180 185 190
Ala?Thr?Pro?Lys?Lys?Asn?Val?Gly?Val?Gln?Gln?Gln?Tyr?Val?Thr?Lys
195 200 205
Ala?Val?Tyr?Pro?Val?Thr?Val?Pro?Ser?Ala?Val?Ala?Ala?Pro?Val?Gln
210 215 220
Ala?Tyr?Val?Ala?Pro?Pro?Glu?Ser?Asp?Val?Thr?Cys?Thr?Thr?Ile?Ser
225 230 235 240
Val?Ala?Asn?Leu?Asp?Gln?Asn?Val?Thr?Glu?Glu?Glu?Leu?Lys?Lys?Ala
245 250 255
Phe?Ser?Gln?Leu?Gly?Glu?Val?Ile?Tyr?Val?Lys?Ile?Pro?Ala?Thr?Lys
260 265 270
Gly?Tyr?Gly?Tyr?Val?Gln?Phe?Lys?Thr?Arg?Pro?Ser?Ala?Glu?Glu?Ala
275 280 285
Val?Gln?Arg?Met?Gln?Gly?Gln?Val?Ile?Gly?Gln?Gln?Ala?Val?Arg?Ile
290 295 300
Ser?Trp?Ser?Lys?Asn?Pro?Gly?Gln?Asp?Gly?Trp?Val?Thr?Gln?Ala?Asp
305 310 315 320
Pro?Asn?Gln?Trp?Asn?Gly?Tyr?Tyr?Gly?Tyr?Gly?Gln?Gly?Tyr?Asp?Ala
325 330 335
Tyr?Ala?Tyr?Gly?Ala?Thr?Gln?Asp?Pro?Ser?Val?Tyr?Ala?Tyr?Gly?Gly
340 345 350
Tyr?Gly?Tyr?Pro?Gln?Tyr?Pro?Gln?Gln?Gly?Glu?Gly?Thr?Gln?Asp?Ile
355 360 365
Ser?Asn?Ser?Ala?Ala?Gly?Gly?Val?Ala?Gly?Ala?Glu?Gln?Glu?Leu?Tyr
370 375 380
Asp?Pro?Leu?Ala?Thr?Pro?Asp?Val?Asp?Lys?Leu?Asn?Ala?Ala?Tyr?Leu
385 390 395 400
Ser?Val?His?Ala?Ser?Ala?Ile?Leu?Gly?Arg?Pro?Met?Trp?Gln?Arg?Thr
405 410 415
Ser?Ser?Leu?Thr?Ser?Gln?Leu?Gly?Lys
420 425
<210>19
<211>1338
<212>DNA
＜213〉Arabidopis thaliana
<400>19
atgcagacac?caaacaacaa?cggttcaaca?gattcagtgt?taccaccaac?atcagccgga 60
acaacaccac?caccaccgtt?gcagcaatca?acaccaccac?cgcagcagca?acaacaacaa 120
cagtggcaac?aacaacaaca?atggatggct?gcgatgcagc?aataccctgc?agctgctatg 180
gctatgatgc?aacaacaaca?gatgatgatg?tatcctcacc?ctcaatacgc?tccttacaat 240
caagctgctt?atcaacagca?tcctcagttt?caatacgctg?cttatcaaca?gcagcagcag 300
caacatcacc?agagtcagca?gcagccacgc?ggtggatctg?gtggtgatga?tgtcaagact 360
ctttgggttg?gtgatcttct?tcattggatg?gatgagactt?atctccatac?ctgtttctct 420
cacaccaatg?aggtttcttc?tgtgaaagtt?atacgcaaca?agcaaacttg?tcaatctgaa 480
ggatatgggt?ttgttgagtt?tctttcacgt?tcagcagctg?aggaagctct?tcagagcttt 540
agcggtgtta?caatgccgaa?cgcggaacag?cctttccgtt?taaactgggc?atctttcagt 600
actggtgaga?aaagagcatc?agagaatggt?cctgacctat?ccatatttgt?tggagatttg 660
gctccagatg?tgagtgatgc?tgtcttgctt?gagacttttg?ctggtagata?tccatctgtc 720
aaaggtgcta?aagttgtgat?tgattccaac?actgggcgtt?ccaaaggtta?cgggtttgtt 780
aggtttggtg?atgagaatga?gcgatcaaga?gctatgacag?aaatgaatgg?tgctttctgt 840
tcaagcaggc?aaatgcgtgt?tggtatcgca?accccgaaaa?gggctgctgc?ttacggccaa 900
caaaatggtt?cacaagctct?tacacttgct?ggtggacatg?gagggaatgg?ttcaatgtct 960
gatggagaat?caaataactc?aacaatattt?gttggcggtc?ttgatgctga?tgttactgaa 1020
gaagacctca?tgcaaccttt?ttccgatttt?ggggaggttg?tttcagtgaa?gatcccagta 1080
gggaaaggat?gtggctttgt?ccaatttgct?aacaggcaaa?gtgctgagga?agccatcggg 1140
aacttgaacg?ggacagtcat?tgggaagaac?actgtccgcc?tttcatgggg?aagaagcccc 1200
aacaaacagt?ggagaagtga?ctctggcaac?caatggaatg?gaggatattc?aagaggtcaa 1260
ggatacaaca?atggatatgc?caatcaggac?tcaaacatgt?acgctactgc?agcggctgca 1320
gtcccgggag?cttcttga 1338
<210>20
<211>445
<212>PRT
＜213〉Arabidopis thaliana
<400>20
Met?Gln?Thr?Pro?Asn?Asn?Asn?Gly?Ser?Thr?Asp?Ser?Val?Leu?Pro?Pro
1 5 10 15
Thr?Ser?Ala?Gly?Thr?Thr?Pro?Pro?Pro?Pro?Leu?Gln?Gln?Ser?Thr?Pro
20 25 30
Pro?Pro?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Trp?Gln?Gln?Gln?Gln?Gln?Trp
35 40 45
Met?Ala?Ala?Met?Gln?Gln?Tyr?Pro?Ala?Ala?Ala?Met?Ala?Met?Met?Gln
50 55 60
Gln?Gln?Gln?Met?Met?Met?Tyr?Pro?His?Pro?Gln?Tyr?Ala?Pro?Tyr?Asn
65 70 75 80
Gln?Ala?Ala?Tyr?Gln?Gln?His?Pro?Gln?Phe?Gln?Tyr?Ala?Ala?Tyr?Gln
85 90 95
Gln?Gln?Gln?Gln?Gln?His?His?Gln?Ser?Gln?Gln?Gln?Pro?Arg?Gly?Gly
100 105 110
Ser?Gly?Gly?Asp?Asp?Val?Lys?Thr?Leu?Trp?Val?Gly?Asp?Leu?Leu?His
115 120 125
Trp?Met?Asp?Glu?Thr?Tyr?Leu?His?Thr?Cys?Phe?Ser?His?Thr?Asn?Glu
130 135 140
Val?Ser?Ser?Val?Lys?Val?Ile?Arg?Asn?Lys?Gln?Thr?Cys?Gln?Ser?Glu
145 150 155 160
Gly?Tyr?Gly?Phe?Val?Glu?Phe?Leu?Ser?Arg?Ser?Ala?Ala?Glu?Glu?Ala
165 170 175
Leu?Gln?Ser?Phe?Ser?Gly?Val?Thr?Met?Pro?Asn?Ala?Glu?Gln?Pro?Phe
180 185 190
Arg?Leu?Asn?Trp?Ala?Ser?Phe?Ser?Thr?Gly?Glu?Lys?Arg?Ala?Ser?Glu
195 200 205
Asn?Gly?Pro?Asp?Leu?Ser?Ile?Phe?Val?Gly?Asp?Leu?Ala?Pro?Asp?Val
210 215 220
Ser?Asp?Ala?Val?Leu?Leu?Glu?Thr?Phe?Ala?Gly?Arg?Tyr?Pro?Ser?Val
225 230 235 240
Lys?Gly?Ala?Lys?Val?Val?Ile?Asp?Ser?Asn?Thr?Gly?Arg?Ser?Lys?Gly
245 250 255
Tyr?Gly?Phe?Val?Arg?Phe?Gly?Asp?Glu?Asn?Glu?Arg?Ser?Arg?Ala?Met
260 265 270
Thr?Glu?Met?Asn?Gly?Ala?Phe?Cys?Ser?Ser?Arg?Gln?Met?Arg?Val?Gly
275 280 285
lle?Ala?Thr?Pro?Lys?Arg?Ala?Ala?Ala?Tyr?Gly?Gln?Gln?Asn?Gly?Ser
290 295 300
Gln?Ala?Leu?Thr?Leu?Ala?Gly?Gly?His?Gly?Gly?Asn?Gly?Ser?Met?Ser
305 310 315 320
Asp?Gly?Glu?Ser?Asn?Asn?Ser?Thr?Ile?Phe?Val?Gly?Gly?Leu?Asp?Ala
325 330 335
Asp?Val?Thr?Glu?Glu?Asp?Leu?Met?Gln?Pro?Phe?Ser?Asp?Phe?Gly?Glu
340 345 350
Val?Val?Ser?Val?Lys?Ile?Pro?Val?Gly?Lys?Gly?Cys?Gly?Phe?Val?Gln
355 360 365
Phe?Ala?Asn?Arg?Gln?Ser?Ala?Glu?Glu?Ala?Ile?Gly?Asn?Leu?Asn?Gly
370 375 380
Thr?Val?Ile?Gly?Lys?Asn?Thr?Val?Arg?Leu?Ser?Trp?Gly?Arg?Ser?Pro
385 390 395 400
Asn?Lys?Gln?Trp?Arg?Ser?Asp?Ser?Gly?Asn?Gln?Trp?Asn?Gly?Gly?Tyr
405 410 415
Ser?Arg?Gly?Gln?Gly?Tyr?Asn?Asn?Gly?Tyr?Ala?Asn?Gln?Asp?Ser?Asn
420 425 430
Met?Tyr?Ala?Thr?Ala?Ala?Ala?Ala?Val?Pro?Gly?Ala?Ser
435 440 445
<210>21
<211>1308
<212>DNA
＜213〉Arabidopis thaliana
<400>21
atgcagacaa?ccaacggctc?agattcgacg?ttggcaactt?ccggagccac?accgccgaat 60
caacaaaccc?ctcctccacc?tcagcagtgg?cagcagcagc?aacagcaaca?gcaacagtgg 120
atggctgcca?tgcaatatcc?accagcggcg?gcgatgatga?tgatgcagca?gcaacagatg 180
ctgatgtatc?ctcatcaata?tgttccgtat?aatcaaggtc?cttatcagca?gcatcatcct 240
cagcttcacc?aatacgggtc?ttatcaacag?caccagcacc?agcaacacaa?ggctattgac 300
cgtggatctg?gagatgatgt?caagactctt?tgggttggtg?atcttcttca?ttggatggat 360
gagacttatc?tccattcttg?cttttctcac?accggcgagg?tttcttctgt?gaaagttata 420
cgtaacaagc?tcacttctca?atcagaaggg?tatgggtttg?ttgagtttct?ttcacgtgct 480
gcagctgaag?aagttcttca?gaactatagt?ggttcagtga?tgccaaactc?ggaccaaccc 540
ttccgtataa?actgggcatc?ttttagtact?ggtgaaaaaa?gagcagtgga?aaatggtcca 600
gacctatctg?tttttgtggg?agacttgtct?ccagatgtca?ctgacgtttt?attgcatgag 660
accttttctg?atagatatcc?ttctgtcaaa?agcgccaaag?ttgtgattga?ttccaacacc 720
ggccggtcca?aaggttatgg?ttttgtgagg?ttcggtgatg?aaaatgagag?gtcaagggct 780
ttgacagaaa?tgaatggagc?ttactgttcg?aacaggcaaa?tgcgtgtagg?tattgcaact 840
cccaaaagag?cgattgctaa?tcagcaacaa?cattcttcac?aagctgtgat?tctggctggt 900
ggacatggat?caaatggttc?catgggttat?ggctcgcagt?ctgatggcga?atcaactaac 960
gcaacaatat?ttgttggcgg?cattgaccct?gatgttattg?atgaagacct?cagacaacct 1020
ttttcccagt?ttggagaggt?tgtttcagtg?aagatcccag?tagggaaagg?atgtggattt 1080
gtccaatttg?ctgacaggaa?gagtgctgaa?gatgctatcg?agagtttgaa?cgggacagtc 1140
atcggcaaga?acactgtcag?actctcctgg?ggacgaagcc?caaacaagca?gtggagagga 1200
gactcagggc?agcagtggaa?tggaggatac?tcacgaggac?atggttacaa?caatggagga 1260
ggatatgcta?accaccacga?ctccaacaac?tatcatgggg?agaattga 1308
<210>22
<211>435
<212>PRT
＜213〉Arabidopis thaliana
<400>22
Met?Gln?Thr?Thr?Asn?Gly?Ser?Asp?Ser?Thr?Leu?Ala?Thr?Ser?Gly?Ala
1 5 10 15
Thr?Pro?Pro?Asn?Gln?Gln?Thr?Pro?Pro?Pro?Pro?Gln?Gln?Trp?Gln?Gln
20 25 30
Gln?Gln?Gln?Gln?Gln?Gln?Gln?Trp?Met?Ala?Ala?Met?Gln?Tyr?Pro?Pro
35 40 45
Ala?Ala?Ala?Met?Met?Met?Met?Gln?Gln?Gln?Gln?Met?Leu?Met?Tyr?Pro
50 55 60
His?Gln?Tyr?Val?Pro?Tyr?Asn?Gln?Gly?Pro?Tyr?Gln?Gln?His?His?Pro
65 70 75 80
Gln?Leu?His?Gln?Tyr?Gly?Ser?Tyr?Gln?Gln?His?Gln?His?Gln?Gln?His
85 90 95
Lys?Ala?Ile?Asp?Arg?Gly?Ser?Gly?Asp?Asp?Val?Lys?Thr?Leu?Trp?Val
100 105 110
Gly?Asp?Leu?Leu?His?Trp?Met?Asp?Glu?Thr?Tyr?Leu?His?Ser?Cys?Phe
115 120 125
Ser?His?Thr?Gly?Glu?Val?Ser?Ser?Val?Lys?Val?Ile?Arg?Asn?Lys?Leu
130 135 140
Thr?Ser?Gln?Ser?Glu?Gly?Tyr?Gly?Phe?Val?Glu?Phe?Leu?Ser?Arg?Ala
145 150 155 160
Ala?Ala?Glu?Glu?Val?Leu?Gln?Asn?Tyr?Ser?Gly?Ser?Val?Met?Pro?Asn
165 170 175
Ser?Asp?Gln?Pro?Phe?Arg?Ile?Asn?Trp?Ala?Ser?Phe?Ser?Thr?Gly?Glu
180 185 190
Lys?Arg?Ala?Val?Glu?Asn?Gly?Pro?Asp?Leu?Ser?Val?Phe?Val?Gly?Asp
195 200 205
Leu?Ser?Pro?Asp?Val?Thr?Asp?Val?Leu?Leu?His?Glu?Thr?Phe?Ser?Asp
210 215 220
Arg?Tyr?Pro?Ser?Val?Lys?Ser?Ala?Lys?Val?Val?Ile?Asp?Ser?Asn?Thr
225 230 235 240
Gly?Arg?Ser?Lys?Gly?Tyr?Gly?Phe?Val?Arg?Phe?Gly?Asp?Glu?Asn?Glu
245 250 255
Arg?Ser?Arg?Ala?Leu?Thr?Glu?Met?Asn?Gly?Ala?Tyr?Cys?Ser?Asn?Arg
260 265 270
Gln?Met?Arg?Val?Gly?Ile?Ala?Thr?Pro?Lys?Arg?Ala?Ile?Ala?Asn?Gln
275 280 285
Gln?Gln?His?Ser?Ser?Gln?Ala?Val?Ile?Leu?Ala?Gly?Gly?His?Gly?Ser
290 295 300
Asn?Gly?Ser?Met?Gly?Tyr?Gly?Ser?Gln?Ser?Asp?Gly?Glu?Ser?Thr?Asn
305 310 315 320
Ala?Thr?Ile?Phe?Val?Gly?Gly?Ile?Asp?Pro?Asp?Val?Ile?Asp?Glu?Asp
325 330 335
Leu?Arg?Gln?Pro?Phe?Ser?Gln?Phe?Gly?Glu?Val?Val?Ser?Val?Lys?Ile
340 345 350
Pro?Val?Gly?Lys?Gly?Cys?Gly?Phe?Val?Gln?Phe?Ala?Asp?Arg?Lys?Ser
355 360 365
Ala?Glu?Asp?Ala?Ile?Glu?Ser?Leu?Asn?Gly?Thr?Val?Ile?Gly?Lys?Asn
370 375 380
Thr?Val?Arg?Leu?Ser?Trp?Gly?Arg?Ser?Pro?Asn?Lys?Gln?Trp?Arg?Gly
385 390 395 400
Asp?Ser?Gly?Gln?Gln?Trp?Asn?Gly?Gly?Tyr?Ser?Arg?Gly?His?Gly?Tyr
405 410 415
Asn?Asn?Gly?Gly?Gly?Tyr?Ala?Asn?His?His?Asp?Ser?Asn?Asn?Tyr?His
420 425 430
Gly?Glu?Asn
435
<210>23
<211>1299
<212>DNA
＜213〉Arabidopis thaliana
<400>23
atggcagacg?tcaagattca?atccgaatcc?gaatcctcgg?attctcatcc?agtggtcgac 60
aatcaaccac?ctcctccgcc?tccgccgccg?caacagccgg?cgaaagaaga?ggagaatcaa 120
ccaaaaacat?ctccgactcc?gccgccacac?tggatgcggt?atccaccaac?ggtgataatc 180
cctcatcaga?tgatgtacgc?gccgccgccg?ttcccacctt?atcatcagta?tccgaatcac 240
caccaccttc?accatcaatc?tcgtggtaat?aagcatcaaa?acgcttttaa?tggtgagaat 300
aaaaccatat?gggttggtga?tttgcatcac?tggatggatg?aggcttatct?taattcttct 360
tttgcttccg?gcgacgagag?agagattgtt?tcggtgaagg?tgattcgtaa?taagaacaat 420
ggtttatcag?aaggatatgg?atttgtggag?tttgagtccc?atgatgtagc?tgataaggtt 480
ttgcgggagt?ttaacgggac?gactatgcca?aatactgacc?aaccttttcg?tttgaactgg 540
gctagtttta?gcaccggtga?gaagcggtta?gagaacaatg?gacctgatct?ctctattttc 600
gtgggggatt?tgtcaccaga?tgtttcggat?aatttgttgc?acgagacctt?ctctgagaag 660
tatccgtcgg?ttaaagctgc?gaaagttgtc?cttgatgcta?atactggtag?gtcaaagggg 720
tatgggtttg?tgaggtttgg?tgatgagaat?gaaaggacca?aagcaatgac?tgagatgaat 780
ggtgttaaat?gttctagtag?agctatgcgc?atcggtcctg?ctaccccgag?gaagactaat 840
ggttatcaac?aacaaggtgg?atacatgccg?aatggtacct?tgacgcgtcc?tgaaggggac 900
ataatgaaca?caacaatatt?tgttggaggg?cttgactcta?gtgtcactga?tgaagactta 960
aagcaacctt?tcaatgaatt?cggggaaata?gtctctgtca?agattcctgt?tggtaaagga 1020
tgcggatttg?ttcagtttgt?taacagacca?aatgcagagg?aggctttgga?gaaactaaat 1080
gggactgtaa?ttggaaaaca?aacagttcgg?ctttcttggg?gacgtaatcc?cgccaataag 1140
cagcctagag?ataagtatgg?aaaccaatgg?gttgatccgt?actatggagg?acagttttac 1200
aatgggtatg?gatacatggt?acctcaacct?gacccgagaa?tgtatcccgc?tgcaccttac 1260
tatccaatgt?acggtggtca?tcagcaacaa?gttagctga 1299
<210>24
<211>432
<212>PRT
＜213〉Arabidopis thaliana
<400>24
Met?Ala?Asp?Val?Lys?Ile?Gln?Ser?Glu?Ser?Glu?Ser?Ser?Asp?Ser?His
1 5 10 15
Pro?Val?Val?Asp?Asn?Gln?Pro?Pro?Pro?Pro?Pro?Pro?Pro?Pro?Gln?Gln
20 25 30
Pro?Ala?Lys?Glu?Glu?Glu?Asn?Gln?Pro?Lys?Thr?Ser?Pro?Thr?Pro?Pro
35 40 45
Pro?His?Trp?Met?Arg?Tyr?Pro?Pro?Thr?Val?Ile?Ile?Pro?His?Gln?Met
50 55 60
Met?Tyr?Ala?Pro?Pro?Pro?Phe?Pro?Pro?Tyr?His?Gln?Tyr?Pro?Asn?His
65 70 75 80
His?His?Leu?His?His?Gln?Ser?Arg?Gly?Asn?Lys?His?Gln?Asn?Ala?Phe
85 90 95
Asn?Gly?Glu?Asn?Lys?Thr?Ile?Trp?Val?Gly?Asp?Leu?His?His?Trp?Met
100 105 110
Asp?Glu?Ala?Tyr?Leu?Asn?Ser?Ser?Phe?Ala?Ser?Gly?Asp?Glu?Arg?Glu
115 120 125
Ile?Val?Ser?Val?Lys?Val?Ile?Arg?Asn?Lys?Asn?Asn?Gly?Leu?Ser?Glu
130 135 140
Gly?Tyr?Gly?Phe?Val?Glu?Phe?Glu?Ser?His?Asp?Val?Ala?Asp?Lys?Val
145 150 155 160
Leu?Arg?Glu?Phe?Asn?Gly?Thr?Thr?Met?Pro?Asn?Thr?Asp?Gln?Pro?Phe
165 170 175
Arg?Leu?Asn?Trp?Ala?Ser?Phe?Ser?Thr?Gly?Glu?Lys?Arg?Leu?Glu?Asn
180 185 190
Asn?Gly?Pro?Asp?Leu?Ser?Ile?Phe?Val?Gly?Asp?Leu?Ser?Pro?Asp?Val
195 200 205
Ser?Asp?Asn?Leu?Leu?His?Glu?Thr?Phe?Ser?Glu?Lys?Tyr?Pro?Ser?Val
210 215 220
Lys?Ala?Ala?Lys?Val?Val?Leu?Asp?Ala?Asn?Thr?Gly?Arg?Ser?Lys?Gly
225 230 235 240
Tyr?Gly?Phe?Val?Arg?Phe?Gly?Asp?Glu?Asn?Glu?Arg?Thr?Lys?Ala?Met
245 250 255
Thr?Glu?Met?Asn?Gly?Val?Lys?Cys?Ser?Ser?Arg?Ala?Met?Arg?Ile?Gly
260 265 270
Pro?Ala?Thr?Pro?Arg?Lys?Thr?Asn?Gly?Tyr?Gln?Gln?Gln?Gly?Gly?Tyr
275 280 285
Met?Pro?Asn?Gly?Thr?Leu?Thr?Arg?Pro?Glu?Gly?Asp?Ile?Met?Asn?Thr
290 295 300
Thr?Ile?Phe?Val?Gly?Gly?Leu?Asp?Ser?Ser?Val?Thr?Asp?Glu?Asp?Leu
305 310 315 320
Lys?Gln?Pro?Phe?Asn?Glu?Phe?Gly?Glu?Ile?Val?Ser?Val?Lys?Ile?Pro
325 330 335
Val?Gly?Lys?Gly?Cys?Gly?Phe?Val?Gln?Phe?Val?Asn?Arg?Pro?Asn?Ala
340 345 350
Glu?Glu?Ala?Leu?Glu?Lys?Leu?Asn?Gly?Thr?Val?Ile?Gly?Lys?Gln?Thr
355 360 365
Val?Arg?Leu?Ser?Trp?Gly?Arg?Asn?Pro?Ala?Asn?Lys?Gln?Pro?Arg?Asp
370 375 380
Lys?Tyr?Gly?Asn?Gln?Trp?Val?Asp?Pro?Tyr?Tyr?Gly?Gly?Gln?Phe?Tyr
385 390 395 400
Asn?Gly?Tyr?Gly?Tyr?Met?Val?Pro?Gln?Pro?Asp?Pro?Arg?Met?Tyr?Pro
405 410 415
Ala?Ala?Pro?Tyr?Tyr?Pro?Met?Tyr?Gly?Gly?His?Gln?Gln?Gln?Val?Ser
420 425 430
<210>25
<211>1281
<212>DNA
＜213〉Arabidopis thaliana
<400>25
atgcagaatc?aaaggcttat?taagcagcaa?caacaacaac?aacaacagca?acatcaacaa 60
gctatgattc?aacaagctat?gatgcaacaa?catccttctc?tttatcatcc?tggtgttatg 120
gctcctcctc?agatggagcc?tttaccaagt?ggaaaccttc?ctcctggttt?tgatccaact 180
acttgccgta?gtgtgtatgc?tggaaacatt?catacgcagg?tcacagagat?tcttcttcaa 240
gagatttttg?caagtactgg?tcctattgaa?agctgtaaac?tcatcagaaa?ggataagtca 300
tcatatggat?ttgttcacta?ctttgatcga?agatgtgcta?gtatggctat?aatgactctt 360
aacggaaggc?atatatttgg?acagcctatg?aaagttaatt?gggcgtatgc?aactggtcaa 420
agggaagata?catcaagtca?tttcaacatt?tttgttggag?atcttagtcc?agaggttact 480
gatgcagcat?tgtttgatag?cttttctgct?tttaacagct?gctcggacgc?aagagtaatg 540
tgggaccaga?aaactggacg?ctcaagaggc?tttggttttg?tttccttccg?taatcagcag 600
gatgctcaaa?ctgccataaa?tgagatgaat?ggtaaatggg?taagtagcag?acagatcaga 660
tgcaactggg?cgacaaaagg?tgctactttt?ggcgaggaca?aacatagctc?tgatggaaaa 720
agtgttgtag?aacttactaa?cggatcttca?gaggatggta?gagagctgtc?aaatgaagat 780
gcccctgaaa?acaatcctca?atttacaact?gtctatgtag?gaaatctctc?tccagaagta 840
actcagcttg?atctacaccg?tctattctat?acccttggtg?ctggagtgat?cgaagaggtc 900
cgtgtccagc?gagacaaagg?gtttggtttt?gtgagatata?acactcatga?cgaggctgct 960
cttgctattc?agatgggcaa?cgctcagcct?ttcctcttta?gcagacagat?aaggtgttcc 1020
tggggaaaca?aaccaactcc?atcaggcaca?gcctcaaacc?cacttccccc?accagccccg 1080
gcatcagtcc?cttctctgtc?tgcaatggac?ctcttagcct?acgagaggca?actggctcta 1140
gccaagatgc?atcctcaggc?tcaacattct?ctgaggcaag?caggtcttgg?agtcaatgtt 1200
gctggaggaa?ctgcagctat?gtatgatggt?ggctatcaga?atgtagctgc?ggcccatcag 1260
cagctcatgt?actatcagta?a 1281
<210>26
<211>426
<212>PRT
＜213〉Arabidopis thaliana
<400>26
Met?Gln?Asn?Gln?Arg?Leu?Ile?Lys?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln
1 5 10 15
Gln?His?Gln?Gln?Ala?Met?Ile?Gln?Gln?Ala?Met?Met?Gln?Gln?His?Pro
20 25 30
Ser?Leu?Tyr?His?Pro?Gly?Val?Met?Ala?Pro?Pro?Gln?Met?Glu?Pro?Leu
35 40 45
Pro?Ser?Gly?Asn?Leu?Pro?Pro?Gly?Phe?Asp?Pro?Thr?Thr?Cys?Arg?Ser
50 55 60
Val?Tyr?Ala?Gly?Asn?lle?His?Thr?Gln?Val?Thr?Glu?Ile?Leu?Leu?Gln
65 70 75 80
Glu?Ile?Phe?Ala?Ser?Thr?Gly?Pro?Ile?Glu?Ser?Cys?Lys?Leu?Ile?Arg
85 90 95
Lys?Asp?Lys?Ser?Ser?Tyr?Gly?Phe?Val?His?Tyr?Phe?Asp?Arg?Arg?Cys
100 105 110
Ala?Ser?Met?Ala?Ile?Met?Thr?Leu?Asn?Gly?Arg?His?Ile?Phe?Gly?Gln
115 120 125
Pro?Met?Lys?Val?Asn?Trp?Ala?Tyr?Ala?Thr?Gly?Gln?Arg?Glu?Asp?Thr
130 135 140
Ser?Ser?His?Phe?Asn?Ile?Phe?Val?Gly?Asp?Leu?Ser?Pro?Glu?Val?Thr
145 150 155 160
Asp?Ala?Ala?Leu?Phe?Asp?Ser?Phe?Ser?Ala?Phe?Asn?Ser?Cys?Ser?Asp
165 170 175
Ala?Arg?Val?Met?Trp?Asp?Gln?Lys?Thr?Gly?Arg?Ser?Arg?Gly?Phe?Gly
180 185 190
Phe?Val?Ser?Phe?Arg?Asn?Gln?Gln?Asp?Ala?Gln?Thr?Ala?Ile?Asn?Glu
195 200 205
Met?Asn?Gly?Lys?Trp?Val?Ser?Ser?Arg?Gln?Ile?Arg?Cys?Asn?Trp?Ala
210 215 220
Thr?Lys?Gly?Ala?Thr?Phe?Gly?Glu?Asp?Lys?His?Ser?Ser?Asp?Gly?Lys
225 230 235 240
Ser?Val?Val?Glu?Leu?Thr?Asn?Gly?Ser?Ser?Glu?Asp?Gly?Arg?Glu?Leu
245 250 255
Ser?Asn?Glu?Asp?Ala?Pro?Glu?Asn?Asn?Pro?Gln?Phe?Thr?Thr?Val?Tyr
260 265 270
Val?Gly?Asn?Leu?Ser?Pro?Glu?Val?Thr?Gln?Leu?Asp?Leu?His?Arg?Leu
275 280 285
Phe?Tyr?Thr?Leu?Gly?Ala?Gly?Val?Ile?Glu?Glu?Val?Arg?Val?Gln?Arg
290 295 300
Asp?Lys?Gly?Phe?Gly?Phe?Val?Arg?Tyr?Asn?Thr?His?Asp?Glu?Ala?Ala
305 310 315 320
Leu?Ala?Ile?Gln?Met?Gly?Asn?Ala?Gln?Pro?Phe?Leu?Phe?Ser?Arg?Gln
325 330 335
Ile?Arg?Cys?Ser?Trp?Gly?Asn?Lys?Pro?Thr?Pro?Ser?Gly?Thr?Ala?Ser
340 345 350
Asn?Pro?Leu?Pro?Pro?Pro?Ala?Pro?Ala?Ser?Val?Pro?Ser?Leu?Ser?Ala
355 360 365
Met?Asp?Leu?Leu?Ala?Tyr?Glu?Arg?Gln?Leu?Ala?Leu?Ala?Lys?Met?His
370 375 380
Pro?Gln?Ala?Gln?His?Ser?Leu?Arg?Gln?Ala?Gly?Leu?Gly?Val?Asn?Val
385 390 395 400
Ala?Gly?Gly?Thr?Ala?Ala?Met?Tyr?Asp?Gly?Gly?Tyr?Gln?Asn?Val?Ala
405 410 415
Ala?Ala?His?Gln?Gln?Leu?Met?Tyr?Tyr?Gln
420 425
<210>27
<211>1260
<212>DNA
＜213〉Arabidopis thaliana
<400>27
atgcagaggt?tgaagcagca?gcagcagcag?caacaagtta?tgatgcagca?agctcttatg 60
cagcaacagt?ctctctacca?tcctggtctc?cttgccccgc?cacagataga?accaatccca 120
agtggaaatc?tcccccctgg?ttttgatcca?agtacttgcc?gcagtgtgta?cgttggaaac 180
atccatattc?aggtgacgga?acctctgctt?caagaggttt?ttgctggcac?tggtcctgta 240
gaaagctgta?aactaattag?gaaagaaaag?tcttcttatg?ggtttgtgca?ctactttgat 300
cgaagatcgg?ctggtcttgc?aatcctttct?ctcaatggaa?ggcatttgtt?tgggcaacct 360
atcaaggtta?actgggctta?tgcgagtggc?cagagggagg?atacatcaag?tcacttcaat 420
atatttgttg?gggatttgag?tccggaggtt?actgatgcaa?tgctgtttac?ttgcttctct 480
gtctacccga?cttgctcgga?tgcaagagtt?atgtgggatc?agaaaactgg?gcgttcaaga 540
ggatttggat?ttgtttcctt?ccgtaaccaa?caggatgccc?agactgcaat?agatgagata 600
actgggaaat?ggcttggttc?caggcagata?cgttgcaact?gggcgacaaa?gggagccact 660
tctggtgagg?acaaacagag?ctctgattcc?aaaagcgtcg?tggaacttac?cagtggtgtc 720
tcggaggatg?gtaaagatac?tactaatggt?gaagctcctg?agaacaatgc?tcagtacaca 780
actgtttacg?tcggtaatct?tgctccagag?gtgtcccagg?ttgatcttca?ccgccacttc 840
cattcccttg?gtgctggggt?catagaggaa?gtccgtgttc?aaagagacaa?aggtttcgga 900
tttgtgagat?actctactca?tgtagaggca?gccctcgcta?ttcagatggg?aaacacacat 960
tcctacctta?gtggcaggca?aatgaagtgt?tcttggggaa?gcaagccaac?tccagcagga 1020
acagcttcaa?acccgcttcc?tccaccagct?cctgcaccaa?tcccgggatt?ctcagcgagt 1080
gatctcttgg?cttacgagag?gcaactagcg?atgagcaaga?tggcaggaat?gaatccgatg 1140
atgcatcacc?cgcagggaca?acatgctttt?aaacaagctg?caatgggagc?cactggttca 1200
aaccaggcaa?tatatgacgg?tggttaccag?aacgcgcagc?agctcatgta?ctaccagtaa 1260
<210>28
<211>419
<212>PRT
＜213〉yeast saccharomyces cerevisiae
<400>28
Met?Gln?Arg?Leu?Lys?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Val?Met?Met?Gln
1 5 10 15
Gln?Ala?Leu?Met?Gln?Gln?Gln?Ser?Leu?Tyr?His?Pro?Gly?Leu?Leu?Ala
20 25 30
Pro?Pro?Gln?Ile?Glu?Pro?Ile?Pro?Ser?Gly?Asn?Leu?Pro?Pro?Gly?Phe
35 40 45
Asp?Pro?Ser?Thr?Cys?Arg?Ser?Val?Tyr?Val?Gly?Asn?Ile?His?Ile?Gln
50 55 60
Val?Thr?Glu?Pro?Leu?Leu?Gln?Glu?Val?Phe?Ala?Gly?Thr?Gly?Pro?Val
65 70 75 80
Glu?Ser?Cys?Lys?Leu?Ile?Arg?Lys?Glu?Lys?Ser?Ser?Tyr?Gly?Phe?Val
85 90 95
His?Tyr?Phe?Asp?Arg?Arg?Ser?Ala?Gly?Leu?Ala?Ile?Leu?Ser?Leu?Asn
100 105 110
Gly?Arg?His?Leu?Phe?Gly?Gln?Pro?Ile?Lys?Val?Asn?Trp?Ala?Tyr?Ala
115 120 125
Ser?Gly?Gln?Arg?Glu?Asp?Thr?Ser?Ser?His?Phe?Asn?Ile?Phe?Val?Gly
130 135 140
Asp?Leu?Ser?Pro?Glu?Val?Thr?Asp?Ala?Met?Leu?Phe?Thr?Cys?Phe?Ser
145 150 155 160
Val?Tyr?Pro?Thr?Cys?Ser?Asp?Ala?Arg?Val?Met?Trp?Asp?Gln?Lys?Thr
165 170 175
Gly?Arg?Ser?Arg?Gly?Phe?Gly?Phe?Val?Ser?Phe?Arg?Asn?Gln?Gln?Asp
180 185 190
Ala?Gln?Thr?Ala?Ile?Asp?Glu?Ile?Thr?Gly?Lys?Trp?Leu?Gly?Ser?Arg
195 200 205
Gln?Ile?Arg?Cys?Asn?Trp?Ala?Thr?Lys?Gly?Ala?Thr?Ser?Gly?Glu?Asp
210 215 220
Lys?Gln?Ser?Ser?Asp?Ser?Lys?Ser?Val?Val?Glu?Leu?Thr?Ser?Gly?Val
225 230 235 240
Ser?Glu?Asp?Gly?Lys?Asp?Thr?Thr?Asn?Gly?Glu?Ala?Pro?Glu?Asn?Asn
245 250 255
Ala?Gln?Tyr?Thr?Thr?Val?Tyr?Val?Gly?Asn?Leu?Ala?Pro?Glu?Val?Ser
260 265 270
Gln?Val?Asp?Leu?His?Arg?His?Phe?His?Ser?Leu?Gly?Ala?Gly?Val?Ile
275 280 285
Glu?Glu?Val?Arg?Val?Gln?Arg?Asp?Lys?Gly?Phe?Gly?Phe?Val?Arg?Tyr
290 295 300
Ser?Thr?His?Val?Glu?Ala?Ala?Leu?Ala?Ile?Gln?Met?Gly?Asn?Thr?His
305 310 315 320
Ser?Tyr?Leu?Ser?Gly?Arg?Gln?Met?Lys?Cys?Ser?Trp?Gly?Ser?Lys?Pro
325 330 335
Thr?Pro?Ala?Gly?Thr?Ala?Ser?Asn?Pro?Leu?Pro?Pro?Pro?Ala?Pro?Ala
340 345 350
Pro?Ile?Pro?Gly?Phe?Ser?Ala?Ser?Asp?Leu?Leu?Ala?Tyr?Glu?Arg?Gln
355 360 365
Leu?Ala?Met?Ser?Lys?Met?Ala?Gly?Met?Asn?Pro?Met?Met?His?His?Pro
370 375 380
Gln?Gly?Gln?His?Ala?Phe?Lys?Gln?Ala?Ala?Met?Gly?Ala?Thr?Gly?Ser
385 390 395 400
Asn?Gln?Ala?Ile?Tyr?Asp?Gly?Gly?Tyr?Gln?Asn?Ala?Gln?Gln?Leu?Met
405 410 415
Tyr?Tyr?Gln
<210>29
<211>1284
<212>DNA
＜213〉Arabidopis thaliana
<400>29
atgcagaatc?cgagactgaa?gcaacatcag?cagcaacaac?aacaacaagc?tatgatgcag 60
caacaagctc?tgatgcagca?acactctctt?taccatcctg?gtgttttggc?tcctcctcag 120
ttagagcctg?ttccaagtgg?aaaccttcct?cctggttttg?atcccagtac?ttgccgtagc 180
gtgtatgttg?gaaacatcca?tacacaggtc?acagagcctt?tgcttcaaga?gatttttaca 240
agcactggcc?ctgttgaaag?cagtaaactc?atcagaaagg?ataagtcatc?atatggattt 300
gttcactact?ttgatcgaag?atccgctgct?ctggctatac?tgtctctgaa?cggaaggcat 360
ctgtttggac?agcctatcaa?agtcaattgg?gcgtatgcca?ctggtcagag?ggaagataca 420
tcaagtcatt?tcaacatttt?tgttggagat?ctcagtccag?aggtcactga?tgcaacatta 480
tatcaaagct?tttctgtctt?ttccagttgt?tcggatgcga?gagttatgtg?ggaccaaaaa 540
actgggcgct?cgagaggctt?tgggtttgtt?tccttccgca?atcaacagga?tgctcaaact 600
gccattaatg?agatgaatgg?taagtggtta?agtagcagac?aaatcagatg?caactgggcc 660
acgaagggcg?ctacttctgg?tgatgataag?ctcagttctg?atggaaaaag?tgttgtggaa 720
cttacaactg?gctcatcaga?ggatggtaaa?gagacattaa?atgaggaaac?acctgaaaat 780
aattctcagt?ttaccactgt?ttatgtggga?aaccttgctc?cagaggtaac?tcagcttgat 840
ctacaccgtt?acttccatgc?tcttggcgct?ggagttattg?aggaggtccg?tgtccaacga 900
gacaaaggct?ttggtttcgt?gagatataac?actcatcccg?aagctgctct?tgctattcag 960
atgggtaaca?ctcagcctta?cctctttaac?agacagataa?agtgctcatg?gggaaacaag 1020
ccaactccac?caggtacagc?ctcaaaccca?cttcccccac?ctgccccagc?tccagttcct 1080
ggtctatctg?cagctgatct?cctaaactat?gagaggcaat?tggcacttag?caagatggca 1140
agtgtgaatg?cgttaatgca?tcaacagggt?caacaccctc?taaggcaggc?tcatggaata 1200
aatgccgctg?gagcaactgc?agccatgtat?gatggtggct?ttcagaatgt?agccgccgca 1260
cagcaactca?tgtactatca?gtaa 1284
<210>30
<211>427
<212>PRT
＜213〉Arabidopis thaliana
<400>30
Met?Gln?Asn?Pro?Arg?Leu?Lys?Gln?His?Gln?Gln?Gln?Gln?Gln?Gln?Gln
1 5 10 15
Ala?Met?Met?Gln?Gln?Gln?Ala?Leu?Met?Gln?Gln?His?Ser?Leu?Tyr?His
20 25 30
Pro?Gly?Val?Leu?Ala?Pro?Pro?Gln?Leu?Glu?Pro?Val?Pro?Ser?Gly?Asn
35 40 45
Leu?Pro?Pro?Gly?Phe?Asp?Pro?Ser?Thr?Cys?Arg?Ser?Val?Tyr?Val?Gly
50 55 60
Asn?Ile?His?Thr?Gln?Val?Thr?Glu?Pro?Leu?Leu?Gln?Glu?Ile?Phe?Thr
65 70 75 80
Ser?Thr?Gly?Pro?Val?Glu?Ser?Ser?Lys?Leu?Ile?Arg?Lys?Asp?Lys?Ser
85 90 95
Ser?Tyr?Gly?Phe?Val?His?Tyr?Phe?Asp?Arg?Arg?Ser?Ala?Ala?Leu?Ala
100 105 110
Ile?Leu?Ser?Leu?Asn?Gly?Arg?His?Leu?Phe?Gly?Gln?Pro?Ile?Lys?Val
115 120 125
Asn?Trp?Ala?Tyr?Ala?Thr?Gly?Gln?Arg?Glu?Asp?Thr?Ser?Ser?His?Phe
130 135 140
Asn?Ile?Phe?Val?Gly?Asp?Leu?Ser?Pro?Glu?Val?Thr?Asp?Ala?Thr?Leu
145 150 155 160
Tyr?Gln?Ser?Phe?Ser?Val?Phe?Ser?Ser?Cys?Ser?Asp?Ala?Arg?Val?Met
165 170 175
Trp?Asp?Gln?Lys?Thr?Gly?Arg?Ser?Arg?Gly?Phe?Gly?Phe?Val?Ser?Phe
180 185 190
Arg?Asn?Gln?Gln?Asp?Ala?Gln?Thr?Ala?Ile?Asn?Glu?Met?Asn?Gly?Lys
195 200 205
Trp?Leu?Ser?Ser?Arg?Gln?Ile?Arg?Cys?Asn?Trp?Ala?Thr?Lys?Gly?Ala
210 215 220
Thr?Ser?Gly?Asp?Asp?Lys?Leu?Ser?Ser?Asp?Gly?Lys?Ser?Val?Val?Glu
225 230 235 240
Leu?Thr?Thr?Gly?Ser?Ser?Glu?Asp?Gly?Lys?Glu?Thr?Leu?Asn?Glu?Glu
245 250 255
Thr?Pro?Glu?Asn?Asn?Ser?Gln?Phe?Thr?Thr?Val?Tyr?Val?Gly?Asn?Leu
260 265 270
Ala?Pro?Glu?Val?Thr?Gln?Leu?Asp?Leu?His?Arg?Tyr?Phe?His?Ala?Leu
275 280 285
Gly?Ala?Gly?Val?Ile?Glu?Glu?Val?Arg?Val?Gln?Arg?Asp?Lys?Gly?Phe
290 295 300
Gly?Phe?Val?Arg?Tyr?Asn?Thr?His?Pro?Glu?Ala?Ala?Leu?Ala?Ile?Gln
305 310 315 320
Met?Gly?Asn?Thr?Gln?Pro?Tyr?Leu?Phe?Asn?Arg?Gln?Ile?Lys?Cys?Ser
325 330 335
Trp?Gly?Asn?Lys?Pro?Thr?Pro?Pro?Gly?Thr?Ala?Ser?Asn?Pro?Leu?Pro
340 345 350
Pro?Pro?Ala?Pro?Ala?Pro?Val?Pro?Gly?Leu?Ser?Ala?Ala?Asp?Leu?Leu
355 360 365
Asn?Tyr?Glu?Arg?Gln?Leu?Ala?Leu?Ser?Lys?Met?Ala?Ser?Val?Asn?Ala
370 375 380
Leu?Met?His?Gln?Gln?Gly?Gln?His?Pro?Leu?Arg?Gln?Ala?His?Gly?Ile
385 390 395 400
Asn?Ala?Ala?Gly?Ala?Thr?Ala?Ala?Met?Tyr?Asp?Gly?Gly?Phe?Gln?Asn
405 410 415
Val?Ala?Ala?Ala?Gln?Gln?Leu?Met?Tyr?Tyr?Gln
420 425
<210>31
<211>735
<212>DNA
＜213〉yeast saccharomyces cerevisiae
<400>31
atggccgctg?aaaaaatctt?gaccccagaa?tctcagttga?agaagtctaa?ggctcaacaa 60
aagactgctg?aacaagtcgc?tgctgaaaga?gctgctcgta?aggctgctaa?caaggaaaag 120
agagccatta?ttttggaaag?aaacgccgct?taccaaaagg?aatacgaaac?tgctgaaaga 180
aacatcattc?aagctaagcg?tgatgccaag?gctgctggtt?cctactacgt?cgaagctcaa 240
cacaagttgg?tcttcgttgt?cagaatcaag?ggtattaaca?agatcccacc?taagccaaga 300
aaggttctac?aattgctaag?attgacaaga?atcaactctg?gtacattcgt?caaagttacc 360
aaggctactt?tggaactatt?gaagttgatt?gaaccatacg?ttgcttacgg?ttacccatcg 420
tactctacta?ttagacaatt?ggtctacaag?agaggtttcg?gtaagatcaa?caagcaaaga 480
gttceattgt?ccgacaatgc?tatcatcgaa?gccaacttgg?gtaagtatgg?tatcttgtcc 540
attgacgatt?tgattcacga?aatcatcact?gttggtccac?acttcaagca?agctaacaac 600
tttttgtggc?cattcaagtt?gtccaaccca?tctggtggtt?ggggtgtccc?aagaaagttc 660
aagcacttta?tccaaggtgg?ttctttcggt?aaccgtgaag?aattcatcaa?caaattggtt 720
aagtccatga?actaa 735
<210>32
<211>244
<212>PRT
＜213〉yeast saccharomyces cerevisiae
<400>32
Met?Ala?Ala?Glu?Lys?Ile?Leu?Thr?Pro?Glu?Ser?Gln?Leu?Lys?Lys?Ser
1 5 10 15
Lys?Ala?Gln?Gln?Lys?Thr?Ala?Glu?Gln?Val?Ala?Ala?Glu?Arg?Ala?Ala
20 25 30
Arg?Lys?Ala?Ala?Asn?Lys?Glu?Lys?Arg?Ala?Ile?Ile?Leu?Glu?Arg?Asn
35 40 45
Ala?Ala?Tyr?Gln?Lys?Glu?Tyr?Glu?Thr?Ala?Glu?Arg?Asn?Ile?Ile?Gln
50 55 60
Ala?Lys?Arg?Asp?Ala?Lys?Ala?Ala?Gly?Ser?Tyr?Tyr?Val?Glu?Ala?Gln
65 70 75 80
His?Lys?Leu?Val?Phe?Val?Val?Arg?Ile?Lys?Gly?Ile?Asn?Lys?Ile?Pro
85 90 95
Pro?Lys?Pro?Arg?Lys?Val?Leu?Gln?Leu?Leu?Arg?Leu?Thr?Arg?Ile?Asn
100 105 110
Ser?Gly?Thr?Phe?Val?Lys?Val?Thr?Lys?Ala?Thr?Leu?Glu?Leu?Leu?Lys
115 120 125
Leu?lle?Glu?Pro?Tyr?Val?Ala?Tyr?Gly?Tyr?Pro?Ser?Tyr?Ser?Thr?Ile
130 135 140
Arg?Gln?Leu?Val?Tyr?Lys?Arg?Gly?Phe?Gly?Lys?Ile?Asn?Lys?Gln?Arg
145 150 155 160
Val?Pro?Leu?Ser?Asp?Asn?Ala?Ile?Ile?Glu?Ala?Asn?Leu?Gly?Lys?Tyr
165 170 175
Gly?Ile?Leu?Ser?Ile?Asp?Asp?Leu?Ile?His?Glu?Ile?Ile?Thr?Val?Gly
180 185 190
Pro?His?Phe?Lys?Gln?Ala?Asn?Asn?Phe?Leu?Trp?Pro?Phe?Lys?Leu?Ser
195 200 205
Asn?Pro?Ser?Gly?Gly?Trp?Gly?Val?Pro?Arg?Lys?Phe?Lys?His?Phe?Ile
210 215 220
Gln?Gly?Gly?Ser?Phe?Gly?Asn?Arg?Glu?Glu?Phe?Ile?Asn?Lys?Leu?Val
225 230 235 240
Lys?Ser?Met?Asn
<210>33
<211>735
<212>DNA
＜213〉yeast saccharomyces cerevisiae
<400>33
atgtccactg?aaaaaatctt?gactcctgaa?tctcaattga?agaagactaa?agctcaacaa 60
aagactgcag?aacaaattgc?tgcagagaga?gctgcccgta?aagccgctaa?caaggaaaaa 120
agagctatta?ttttggaaag?aaacgccgct?taccaaaagg?aatacgaaac?tgctgaaaga 180
aacatcattc?aagctaagcg?tgatgccaag?gctgctggtt?cctactacgt?cgaagctcaa 240
cacaagttgg?tcttcgttgt?cagaatcaag?ggtattaaca?agattccacc?taagccaaga 300
aaggttctac?aattgctaag?attgacaaga?atcaactctg?gtacattcgt?caaagttacc 360
aaggctactt?tggaactatt?gaagttgatt?gaaccatacg?ttgcttacgg?ttacccatcc 420
tactctacta?ttagacaatt?ggtctacaag?agaggtttcg?gtaagatcaa?caagcaaaga 480
gttccattgt?ccgacaatgc?tatcatcgaa?gccaacttgg?gtaagtatgg?tatcttgtcc 540
attgacgatt?tgattcacga?aatcatcact?gttggtccac?acttcaagca?agctaacaac 600
tttttgtggc?cattcaagtt?gtccaaccca?tctggtggtt?ggggtgtccc?aagaaagttc 660
aagcatttca?tccaaggtgg?ttctttcggt?aaccgtgaag?aattcatcaa?taaattggtt 720
aaggctatga?actaa 735
<210>34
<211>244
<212>PRT
＜213〉yeast saccharomyces cerevisiae
<400>34
Met?Ser?Thr?Glu?Lys?Ile?Leu?Thr?Pro?Glu?Ser?Gln?Leu?Lys?Lys?Thr
1 5 10 15
Lys?Ala?Gln?Gln?Lys?Thr?Ala?Glu?Gln?Ile?Ala?Ala?Glu?Arg?Ala?Ala
20 25 30
Arg?Lys?Ala?Ala?Asn?Lys?Glu?Lys?Arg?Ala?Ile?Ile?Leu?Glu?Arg?Asn
35 40 45
Ala?Ala?Tyr?Gln?Lys?Glu?Tyr?Glu?Thr?Ala?Glu?Arg?Asa?Ile?Ile?Gln
50 55 60
Ala?Lys?Arg?Asp?Ala?Lys?Ala?Ala?Gly?Ser?Tyr?Tyr?Val?Glu?Ala?Gln
65 70 75 80
His?Lys?Leu?Val?Phe?Val?Val?Arg?Ile?Lys?Gly?Ile?Asn?Lys?Ile?Pro
85 90 95
Pro?Lys?Pro?Arg?Lys?Val?Leu?Gln?Leu?Leu?Arg?Leu?Thr?Arg?Ile?Asn
100 105 110
Ser?Gly?Thr?Phe?Val?Lys?Val?Thr?Lys?Ala?Thr?Leu?Glu?Leu?Leu?Lys
115 120 125
Leu?Ile?Glu?Pro?Tyr?Val?Ala?Tyr?Gly?Tyr?Pro?Ser?Tyr?Ser?Thr?Ile
130 135 140
Arg?Gln?Leu?Val?Tyr?Lys?Arg?Gly?Phe?Gly?Lys?Ile?Asn?Lys?Gln?Arg
145 150 155 160
Val?Pro?Leu?Ser?Asp?Asn?Ala?Ile?Ile?Glu?Ala?Asn?Leu?Gly?Lys?Tyr
165 170 175
Gly?Ile?Leu?Ser?Ile?Asp?Asp?Leu?Ile?His?Glu?Ile?Ile?Thr?Val?Gly
180 185 190
Pro?His?Phe?Lys?Gln?Ala?Asn?Asn?Phe?Leu?Trp?Pro?Phe?Lys?Leu?Ser
195 200 205
Asn?Pro?Ser?Gly?Gly?Trp?Gly?Val?Pro?Arg?Lys?Phe?Lys?His?Phe?Ile
210 215 220
Gln?Gly?Gly?Ser?Phe?Gly?Asn?Arg?Glu?Glu?Phe?Ile?Asn?Lys?Leu?Val
225 230 235 240
Lys?Ala?Met?Asn
<210>35
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>35
aaaaagcagg?cttaatgcag?caaccaccgt?caaacgcc 38
<210>36
<211>37
<212>DNA
<213>Artificial?Sequence
<220>
<223>primer
<400>36
agaaagctgg?gtttcactga?cgttgctgct?gatagtt 37
<210>37
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>37
aaaaagcagg?cttaatgcag?acaccaaaca?acaacggt 38
<210>38
<211>37
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>38
agaaagctgg?gtttcaagaa?gctcccggga?ctgcagc 37
<210>39
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>39
aaaaagcagg?cttaatgcag?acaaccaacg?gctcagat 38
<210>40
<211>37
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>40
agaaagctgg?gtttcaattc?tccccatgat?agttgtt 37
<210>41
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>41
aaaaagcagg?cttaatggca?gacgtcaaga?ttcaatcc 38
<210>42
<211>37
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>42
agaaagctgg?gtttcagcta?acttgttgct?gatgacc 37
<210>43
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>43
aaaaagcagg?cttaatggca?gacgtcaagg?ttcaatcc 38
<210>44
<211>37
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>44
agaaagctgg?gtttcagcta?acttgttgct?gatgacc 37
<210>45
<211>38
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>45
aaaaagcagg?cttaatgcag?aatcaaaggc?ttattaag 38
<210>46
<211>37
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>46
agaaagctgg?gttttactga?tagtacatga?gctgctg 37
<210>47
<211>34
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>47
aaaaagcagg?cttaatgtcc?actgaaaaaa?tctt 31
<210>48
<211>33
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>48
agaaagctgg?gttttagttc?atagccttaa?cca 33
<210>49
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>49
aatctgtgtc?gacgtacttc 20
<210>50
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>50
agaagtacat?aggatgggtc 20
<210>51
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>51
aaaaattgtc?gacgtacttc 20
<210>52
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>52
aaaggaagtt?atcacaattg 20
<210>53
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>53
ceagcatcta?tgtctgcaac 20
<210>54
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>54
cgtatctgga?gtagtatttc 20
<210>55
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>55
gcaaggtata?caaagcagaa 20
<210>56
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>56
tcatcctttt?tcttctctgc 20
<210>57
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>57
gacacgatga?agttggatat 20
<210>58
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>58
tgactgtcaa?atcatcactg 20
<210>59
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>59
cagtataaaa?atgtctgaat 20
<210>60
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>60
tggttgatta?tttcttcttc 20
<210>61
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>61
atcaacgtca?taatgtccac 20
<210>62
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>62
taccagagtt?gattcttgtc 20
<210>63
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>63
acctaaagaa?accatgtcag 20
<210>64
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>64
tatcaaggtt?gtacgtttcg 20
<210>65
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>65
atgtacagtc?taagtcaagg 20
<210>66
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>66
gactaaagtg?aacagcaatg 20
<210>67
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>67
gagaatggca?atatttcaag 20
<210>68
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>68
tgttcttctt?cttccattac 20
<210>69
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>69
tggacccaca?taatccaatt 20
<210>70
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>70
tttcgaacat?tacctcacac 20
<210>71
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>71
gtggatggtc?ttttagaaga 20
<210>72
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>72
aactcctcga?aacttaaacg 20
<210>73
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>73
tattgagacc?ttcttccaag 20
<210>74
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>74
aagattttac?cggaaacgtg 20
<210>75
<211>22
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>75
gacgataaaa?agaaatttgg?tg 22
<210>76
<211>19
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>76
ctcaaagcgt?tgttgaaag 19
<210>77
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>77
gagagaggtc?attagtatta 20
<210>78
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>78
ttttctaata?acagggaacc 20
<210>79
<211>23
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>79
gtttaataga?aaaagaagag?gag 23
<210>80
<211>23
<212>DNA
<213>Artificial?Sequence
<220>
<223>primer
<400>80
tagttcatca?actaaaaaca?tgg 23
<210>81
<211>19
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>81
agctgtccca?agtgttcaa 19
<210>82
<211>19
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>82
acccttacca?ccgaatttc 19
<210>83
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>83
gttgggatat?ttttggttgg 20
<210>84
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>84
aaaggaacgt?ccttcaattc 20
<210>85
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>85
aacaagctgt?tcaggttaga 20
<210>86
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>86
ggtttgtgat?tatcatcagg 20
<210>87
<211>21
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>87
aattaaagat?cacaatggcc?g 21
<210>88
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>88
cttggtaact?ttgacgaatg 20
<210>89
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>89
cagaaaagct?ggtgttcaag 20
<210>90
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>90
tgattctgca?tcgtggtttc 20
<210>91
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>91
ttgattaaga?actccaaagc 20
<210>92
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>92
tcttctcaag?acacgtaatc 20
<210>93
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>93
agatgaggtt?gaagcaatag 20
<210>94
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>94
caagggcaat?ttccttattg 20
<210>95
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>95
taagactaag?caacaatgcc 20
<210>96
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>96
aaacccaact?tgtagacttg 20
<210>97
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>97
gcatctcata?atatgtctgc 20
<210>98
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>98
ttgttgctaa?gactgtagag 20
<210>99
<211>22
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>99
caaatccatt?tcaaaatata?gg 22
<210>100
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>100
ctcctcctat?ctaaaaaacc 20
<210>101
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>101
aagaagagtt?ggtaagcaag 20
<210>102
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>102
caccgttttt?gaatgtgatg 20
<210>103
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>103
agcgtaatac?gaaagatgag 20
<210>104
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>104
agcttcgtta?ttcaagggat 20
<210>105
<211>22
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>105
gtatcataaa?cattcaacaa?tg 22
<210>106
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>106
cggatctgtt?gtttattctc 20
<210>107
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>107
aaacaaagtt?tgatcgcctc 20
<210>108
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>108
tcgtgctcaa?acatttcttc 20
<210>109
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>109
aaaatgacgg?ataatccacc 20
<210>110
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>110
ttcaaagtct?ttagcacacc 20
<210>111
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>111
caatccatca?tgggaaaatc 20
<210>112
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>112
cttggacgac?aaaatagtgt 20
<210>113
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>113
aggacttcaa?tttccatgtc 20
<210>114
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>114
agtgtcatct?ccacaatttg 20
<210>115
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>115
gaaaacgata?agggccaatt 20
<210>116
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>116
cgttctttaa?caaaccatcg 20
<210>117
<211>22
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>117
taccaaatga?aacgctttaa?tg 22
<210>118
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>118
tcttcatgga?aagagtctag 20
<210>119
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>119
atggaatgag?tactttagcg 20
<210>120
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>120
cttcatttcc?gagtttttgg 20
<210>121
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>121
aatagaaaat?cggcttctgc 20
<210>122
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>122
tatttgatca?ttggggttgc 20
<210>123
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>123
gattgaagac?atttgatgcg 20
<210>124
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>124
tcgccactaa?ctctatttac 20
<210>125
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>125
accatttcag?gtacaatgtc 20
<210>126
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>126
cttcggaaat?atcgaattcc 20
<210>127
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>127
ctgaaacgat?accaacaatg 20
<210>128
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>128
tttgtggttt?aggcaatacc 20
<210>129
<211>22
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>129
atacaaaagt?atacaacatg?cc 22
<210>130
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>130
tttccaagaa?atcttcgacc 20
<210>131
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>131
ctttactgcg?aagataaagg 20
<210>132
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>132
gccactataa?tctgttgttg 20
<210>133
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>133
tcaaaactac?ggctcatttg 20
<210>134
<211>20
<212>DNA
＜213〉artificial series
<220>
<223>primer
<400>134
tgaacaaaag?actcaatccg 20

Claims (19)

1, coding is by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have an active protein DNA of imparting boron-tolerance.
2, coding is by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the protein that constitutes of the aminoacid sequence shown in 30 and have the active DNA of imparting boron-tolerance.
3, by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27, or the imparting boron-tolerance gene DNA of the base sequence shown in 29 or its complementary sequence formation.
4,, or disappearance in the base sequence shown in 29, replace or added that the base sequence of 1 or several bases constitutes and coding has the active protein DNA of imparting boron-tolerance by sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27.
5, under stringent condition, hybridize and encode with the DNA of claim 3 record and have the active protein DNA of imparting boron-tolerance.
6, by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or the aminoacid sequence shown in 30 constitute have the active protein of imparting boron-tolerance.
7, by sequence number 2,4,6,8,10,12,14,16,18,20,22,24,26,28, or disappearance in the aminoacid sequence shown in 30, replace or added that the amino acid whose aminoacid sequences of 1 or several constitute, and have the active protein of imparting boron-tolerance.
8, the DNA that contains each record of claim 1～5, and can express the recombinant vectors of proteins imparting boron-tolerance.
9, import the recombinant vectors of claim 8 record, and expressed the proteins imparting boron-tolerance transformant.
10, according to the transformant of claim 9 record, it is characterized in that transformant is a yeast.
11, according to the transformant of claim 9 record, it is characterized in that transformant is a plant.
12, the screening method of imparting boron-tolerance gene, it is characterized in that using gene library, disappearance YNL275w gene, transform the YNL275w that does not express YNL275w and destroy yeast, in containing the substratum of boric acid, cultivate gained and transform YNL275w destruction yeast, measure and estimate this conversion YNL275w and destroy zymic imparting boron-tolerance activity.
13, the screening method of imparting boron-tolerance gene, it is characterized in that suppressing with the specificity to montage that is produced by boric acid is target, measures and estimate the enhancing degree of montage efficient.
14, according to the screening method of the imparting boron-tolerance gene of claim 13 record, it is characterized in that making analyte in yeast cell, to express, in the presence of boric acid, the cultivation analyte is expressed, and measures and estimates as the enhancing degree of montage efficient with the improvement degree that the specificity to montage that is produced by boric acid of the gene that contains intron in the yeast suppresses.
15, according to the screening method of the imparting boron-tolerance gene of claim 14 record, the gene that it is characterized in that containing in the yeast intron is the RPL7B gene in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genome.
16, the DNA of each record of claim 1～5 is as the purposes of imparting boron-tolerance gene.
17, the DNA of each record of claim 1～5 is used to prepare plant or the zymic purposes that has been given the boric acid resistance.
18, the protein of claim 6 or 7 records is as having the active proteinic purposes of imparting boron-tolerance.
19, the protein of claim 6 or 7 records is used to prepare plant or the zymic purposes that has been given the boric acid resistance.
CN200580007305.4A 2004-03-15 2005-03-15 Proteins imparting boron-tolerance and genes thereof Expired - Fee Related CN1930292B (en)

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JP2004073324 2004-03-15
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JPWO2006098464A1 (en) 2005-03-14 2008-08-28 リンク・ジェノミクス株式会社 Diagnosis of prostate cancer
JP6202832B2 (en) * 2012-03-08 2017-09-27 国立研究開発法人農業・食品産業技術総合研究機構 Environmental stress-tolerant plant having high seed yield and its production method
TR201921881A1 (en) * 2019-12-26 2021-07-26 Akdeniz Ueniversitesi BORA RESISTANCE GENES SYNTHETIC FROM mRNA ISOLATED FROM PUCCINELLIA DISTANS PLANT
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CA2672025A1 (en) * 1998-08-04 2000-02-17 Cropdesign N.V. Genes involved in tolerance to environmental stress
EP1033405A3 (en) * 1999-02-25 2001-08-01 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
CA2420555C (en) * 2000-08-24 2012-10-23 Jeffrey F. Harper Stress-regulated genes of plants, transgenic plants containing same, and methods of use
US20060150283A1 (en) * 2004-02-13 2006-07-06 Nickolai Alexandrov Sequence-determined DNA fragments and corresponding polypeptides encoded thereby

Non-Patent Citations (1)

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Title
无: "Arabidopsis thaliana RNA-binding protein 47", 《NCBI ACCESSION NM_103643》 *

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JPWO2005087928A1 (en) 2008-01-31
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CA2559093A1 (en) 2005-09-22
EP1731610A1 (en) 2006-12-13
CN1930292B (en) 2014-04-02
US7666678B2 (en) 2010-02-23
US20070079394A1 (en) 2007-04-05
WO2005087928A1 (en) 2005-09-22
JP4504365B2 (en) 2010-07-14
EP1731610A4 (en) 2008-06-18

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